FLx800™

FLx800™
Multi-Detection Microplate Reader
FLx800™
Operator’s Manual
FLx800™
Multi-Detection Microplate Reader, All Models
Operator’s Manual
July 2011
 2011
Part Number 7081000
Revision J
BioTek® Instruments, Inc.
ii | Preface
Notices
BioTek Instruments, Inc.
Highland Park, P.O. Box 998
Winooski, Vermont 05404-0998 USA
All Rights Reserved
© 2011, BioTek Instruments, Incorporated. No part of this publication may be
reproduced, transcribed, or transmitted in any form, or by any means electronic or
mechanical, including photocopying and recording, for any purpose other than the
purchaser’s use without written permission of BioTek Instruments, Inc.
Trademarks
BioTek® is a registered trademark and FLx800TM is a trademark of BioTek
Instruments, Inc.
Microsoft®, Windows®, and Excel® are either registered trademarks or trademarks
of Microsoft Corporation in the United States and/or other countries.
All other trademarks are the property of their respective holders.
Restrictions and Liabilities
Information in this document is subject to change and does not represent a
commitment by BioTek. Changes made to the information in this document will be
incorporated in new editions of the publication. BioTek assumes no responsibility
for the use or reliability of software or equipment that is not supplied by BioTek or
its affiliated dealers.
BioTek Instruments, Inc.
Contents |
Contents
Preface ............................................................................................ i
Notices ....................................................................................... ii
BioTek Instruments, Inc. ........................................................ ii
All Rights Reserved.................................................................. ii
Trademarks ............................................................................ ii
Restrictions and Liabilities ........................................................ ii
Contents .................................................................................... iii
Contact Information ................................................................... vii
BioTek Instruments, Inc. ...................................................... vii
Customer Service and Sales ................................................... vii
Service/TAC ......................................................................... vii
European Coordination Center/Authorized European Representativevii
Document Conventions ............................................................... viii
Revision History .......................................................................... ix
Intended Use Statement ............................................................. xii
Quality Control........................................................................... xii
Warranty and Product Registration ............................................... xii
Repackaging and Shipping........................................................... xii
Warnings ..................................................................................xiii
Hazards ....................................................................................xiii
Precautions .............................................................................. xiv
CE Mark ................................................................................... xvi
Directive 2004/108/EC: Electromagnetic Compatibility .............. xvi
Directive 73/23/EEC Low Voltage (Safety) ............................... xvi
Directive 2002/96/EC: Waste Electrical and Electronic Equipmentxvii
Directive 98/79/EC: In Vitro Diagnostics ................................. xvii
Electromagnetic Interface and Susceptibility ................................. xvii
USA FCC CLASS A ................................................................ xvii
Canadian Department of Communications Class A .................... xvii
Safety Symbols ........................................................................ xviii
Chapter 1: Introduction.................................................................. 1
Product Overview ......................................................................... 2
Hardware Features ....................................................................... 3
Software Features ........................................................................ 4
Package Contents ......................................................................... 5
Optional Accessories ..................................................................... 5
Technical Specifications ................................................................ 6
Microplates ............................................................................. 6
Sensitivity .............................................................................. 6
Optical ................................................................................... 7
Incubation .............................................................................. 7
ELx808 Operator’s Manual
iii
iv | Preface
Environmental ........................................................................ 7
Physical ................................................................................. 7
Dispense/Read (Optional Reagent Dispensing) ............................ 8
Chapter 2: Instrument Description ................................................. 9
Principles of Operation ................................................................ 10
External Components.................................................................. 11
External Components for the Dispenser Module.............................. 13
Internal Components for Modules with Dispense Capability .............. 15
Excitation and Emission Filter Wheels ........................................... 17
Filter Position Requirements for Dispense/Read Assays .............. 19
Configuration for Luminescence Measurements ......................... 19
Top Optical Probe Adjustment ...................................................... 20
Lamp Assembly .......................................................................... 22
Chapter 3: Installation ................................................................. 25
Unpacking and Inspection ........................................................... 26
Unpacking and Inspecting the Dispenser Module ............................ 30
Select the Operating Environment ................................................ 34
Connect Power ........................................................................... 34
Install the Printer ....................................................................... 35
Printers ........................................................................... 35
Turn on the Reader and Run a System Test ................................... 35
Check/Adjust the Filter Tables ..................................................... 36
Install Software/Connect to Computer (Optional) ........................... 38
Attach the Cable ................................................................... 38
Install Gen5 Software on the Host Computer ............................ 38
Establish Communication ....................................................... 38
Change the Baud Rate ........................................................... 39
Configuring Global Default Options .......................................... 40
Setting Up the Dispenser Module ............................................. 40
Before Repackaging the Instrument .............................................. 42
Chapter 4: Operation .................................................................... 45
Getting Started with Gen5 ........................................................... 46
Introduction .............................................................................. 47
FLx800 Front Panel ..................................................................... 47
The Keypad .......................................................................... 48
System Startup .......................................................................... 49
Main Menu ................................................................................ 50
DEFINE ..................................................................................... 51
Selecting an Assay to Define................................................... 52
Editing the Assay Name ......................................................... 52
Define METHOD .................................................................... 53
Define MAP ........................................................................... 62
Define FORMULA ................................................................... 74
Define CURVE ....................................................................... 86
Panel Assays ........................................................................ 89
READ ........................................................................................ 91
Selecting an Assay to Run ...................................................... 92
BioTek Instruments, Inc.
Contents |
Run-Time Prompts ................................................................ 92
Beginning the Plate Read ....................................................... 94
REPORT .................................................................................... 95
UTILITY .................................................................................... 98
Setting the Date and Time...................................................... 98
Specifying Data Output and Reporting Options .......................... 99
Selecting Read Options ........................................................ 100
Chapter 5: Instrument Qualification ........................................... 101
Overview................................................................................. 102
IQ/OQ/PQ ............................................................................... 102
Recommended Qualification Schedule ......................................... 104
System and Checksum Tests ..................................................... 105
Corners, Sensitivity, and Linearity Tests ...................................... 107
Required Materials............................................................... 108
Solution Preparation ............................................................ 109
Procedure – Gen5 ............................................................... 110
Procedure – Keypad ............................................................ 111
Pipette Map for Sodium Fluorescein Tests ............................... 112
Results Analysis .................................................................. 113
Troubleshooting .................................................................. 114
Gen5 Protocol Reading Parameters ........................................ 115
Onboard Assay Parameters................................................... 116
Using Methylumbelliferone.................................................... 117
Dispense Accuracy & Precision Test ............................................ 123
Required Materials............................................................... 123
Test Solutions ..................................................................... 124
Test Setup ......................................................................... 125
Procedure........................................................................... 125
Results Analysis .................................................................. 127
Gen5 Test Protocols ............................................................. 127
Chapter 6: Preventive Maintenance ............................................ 133
Recommended Maintenance Schedule ......................................... 134
Overview............................................................................ 134
Dispenser Module ................................................................ 134
Schedule ............................................................................ 134
Warnings & Precautions ............................................................ 136
Cleaning Exposed Surfaces ........................................................ 137
Inspecting/Cleaning Excitation and Emission Filters ...................... 138
Flushing/Purging the Fluid Path .................................................. 139
Running a Dispense Protocol (Optional) ...................................... 140
Emptying/Cleaning the Tip Prime Trough ..................................... 141
Cleaning the Supply Bottles ....................................................... 141
Cleaning the Priming Plate ......................................................... 141
Cleaning the Internal Components.............................................. 142
Required Materials............................................................... 143
Removing the Reader’s Cover ............................................... 144
Cleaning the Optical Probe(s) ............................................... 145
ELx808 Operator’s Manual
v
vi | Preface
Removing/Cleaning the Internal Dispense Tube
and Injector Head ............................................................... 148
Cleaning the Internal Instrument Surface ............................... 150
Reassembling the Components ............................................. 151
Performance Check.............................................................. 151
Appendix A: Decontamination .................................................... 153
Purpose .................................................................................. 154
Required Materials.................................................................... 155
Procedure for Models without the Dispenser Module...................... 156
Routine Procedure for Models with the Dispenser Module .............. 157
Clean Exposed Surfaces ....................................................... 157
Decontaminate the Fluid Lines .............................................. 158
Rinse the Fluid Lines ............................................................ 158
Clean the Internal Tubing and Injector Head ........................... 159
Decontaminate/Rinse the Tip Priming Trough and Priming Plate 159
Alternate Procedure for Models with the Dispenser Module ............ 159
Appendix B: Troubleshooting & Error Codes ............................... 163
Product Support & Service ......................................................... 164
Returning Instruments for Service/Repair .................................... 164
Contacting BioTek for Applications Support .................................. 164
Error Codes ............................................................................. 165
Fatal Errors ........................................................................ 165
General Errors .................................................................... 166
Appendix C: Sample Reports ...................................................... 181
BioTek Instruments, Inc.
Contact Information |
Contact Information
BioTek Instruments, Inc.
Highland Park, P.O. Box 998
Winooski, Vermont 05404-0998 USA
Customer Service and Sales
Internet:
www.biotek.com
Phone:
888-451-5171 (toll free in the U.S.)
802-655-4740 (outside the U.S.)
Fax:
802-655-7941
E-Mail:
customercare@biotek.com
Service/TAC
Phone:
800-242-4685 (toll free in the U.S.)
802-655-4740 (outside the U.S.)
Fax:
802-654-0638
E-Mail:
tac@biotek.com
European Coordination Center/Authorized European
Representative
BioTek Instruments GmbH
Kocherwaldstrasse 34
D-74177 Bad Friedrichshall
Germany
Internet:
www.biotek.de
Phone:
+49 (0) 7136-9680
Fax:
+49 (0) 7136-968-111
E-Mail:
info@biotek.de
ELx808 Operator’s Manual
vii
viii | Preface
Document Conventions
This icon calls attention to important safety notes.
Warning!
A Warning indicates the potential for bodily harm and
tells you how to avoid the problem.
Caution
A Caution indicates potential damage to the instrument
and tells you how to avoid the problem.
Note:
Bold text is primarily used for emphasis.
italic
Topics that apply only to specific reader models are
preceded by a notice in italics, for example: Applies only to
FLx800 models with injectors.

This icon calls attention to important information.
BioTek Instruments, Inc.
Revision History |
ix
Revision History
Rev
Date
Changes
A
12/99
Release to Production.
B
7/00
Added Computer Control by KC4 info. Corrected typo on pg 3-17. Corrected optical specs
on the emission range. Explained each function on the transformation function page. Added
metric physical dimensions. Added hardware warranty. Added memory error. Added Power
Supply info in specifications.
C
1/01
Added incubation and shaking information.
D
9/02
Updated contact information (pages iii and B-3).
Added reagent-dispensing (Dispenser Module) feature information as follows:
•
Revised Intended Use Statement (page x)
•
Updated Chapter 1
•
Added descriptions and illustrations to Chapter 2
•
Added unpacking instructions and illustrations to Chapter 3
•
Updated IQ/PQ/OQ verification schedule in Chapter 5
•
Added decontamination and cleaning information to Appendix A
•
Added error codes to Appendix B
• Updated computer control interface information in Appendix C
Modified text on page 1-3, Model FLx800TBEP, second bullet: “To effectively read (added
‘96-well’) microplates using the Top probe, a plate carrier adapter is (changed
‘recommended’ to ‘required.’).”
Added text to page 2-11, item 2 clarifying action of Emission filter wheel.
Clarified range (0 to number of undefined well locations remaining on a plate) when
defining the number of sample groups on a plate (page 4-28).
Added note to page 5-7 advising FLx800TBEP (PCR model) operators to use the BioTek
Plate Carrier Adapter (PN 7080522) to raise the microplate closer to the top probe, to
effectively read 96-well microplates using the top probe.
Replaced “Molecular Probes part no. S-9089” with “1 mg vial, BioTek part no. 98155” in
Required Materials list (page 5-7).
Changed the recommended sensitivity from 50 to 80 for conducting the Corners Plate Test.
Added note to always specify the “Bottom” probe unless the reader only has a “Top” probe
(page 5-9).
Added the recommended sensitivity of 100 for conducting the Sodium Fluorescein Serial
Dilution Test. Added note to always specify the “Bottom” probe unless the reader only has
a “Top” probe (page 5-10).
Updated the index.
E
2/03
Updated E-mail addresses in Notices (page iii) and Appendix B (page B-3).
Added 65ºC specifications for FLx800Ti model – top probe with extended incubation (pages
1-3 and 1-7).
Clarified power consumption specifications: Max 100 Watts for 50°C incubator units; max 130
Watts for FLx800Ti or 65°C incubator units (page 1-7).
Modified inlet and outlet tube callouts in Figure 2-3b (page 2-7).
Changed Emission Filter sensitivity specifications from "530/25" to "528/20" (page 1-6).
Changed Emission Filter type from "530/20" to "528/20" in Figure 2-4b (page 2-10).
ELx808 Operator’s Manual
x | Preface
Rev
Date
(E)
Changes
Changed "LAMBDA" to "FILTER" in Figure 3-6 (page 3-16).
Removed reference to internal bar code scanner option under Enter Plate ID (page 4-49).
Clarified descriptions of inlet and outlet tubes (items 4 and 5, page 3-17).
Updated Verification Schedule and IQ/PQ/OQ requirements (pages 5-3 and 5-4).
Corners Test Procedure note: Added recommendation to ensure that the Top probe height
is adjusted for the plate being used (page 5-9).
Changed Emission Filter type used in Corners Test Procedure from 530/25 nm
to 528/20 nm (page 5-9).
Accuracy and Precision Tests Procedure: Added note to contact the Technical Assistance
Center if testing fails (page 5-18).
Added error codes 1503, 1505, 1506, and 1507 to Incubation Operational Errors (p. B-10).
Updated computer control commands to reflect FLx800Ti model specifications (Appendix
C, pages C-19 and C-31).
F
10/03
Updated Intended Use statement to distinguish between the European Union and all other
jurisdictions. Updated Warranty to include BioTek’s current warranty statement.
Chapter 1, changed Dispense Volume Range from ‘5-200’ to ‘5-1000.’
Chapter 4, changed Delay Before Samples range from ‘100 to 2550’ to ‘10 to 2550.’
Clarified the bleach dilution solutions for Decontamination.
G
8/04
Updated Safety Symbols in Preface (pages x and xi).
Updated PMT performance specifications (Chapter 1).
Updated decontamination procedure. Added instructions for periodic cleaning of the internal
read chamber. (Appendix A).
H
5/06
Added/Modified instructions throughout to support Gen5™.
Removed instructions for repositioning the injector head for models with the external
dispenser module. Added information for configuring the EX/EM filter wheels for
luminescence measurements. Simplified the installation steps for the Dispenser Module.
Added a caution when removing the microplate carrier shipping bracket; do not raise the
carrier from the instrument surface.
In the IQ-OQ-PQ Verification Schedule, removed the step to inspect/clean wavelength filters
(moved them to the new Maintenance Schedule). Removed the requirement to perform the
liquid tests during Installation Qualification (liquid tests are performed during OQ/PQ only).
Added a sample System Test report to Chapter 5, Instrument Qualification.
Simplified the steps for creating the Titration Dyes for the Corners and Sensitivity Tests.
Simplified the steps for performing the serial dilutions for the Sensitivity Test.
Increased the microplate options for performing the Corners and Sensitivity Tests, and
provided specific instructions for using each.
Modified the Dispense Accuracy and Precision Test procedure to use just one microplate for
all three dispense volumes. Added a worksheet for recording Delta OD values, dispense
weights, and pass/fail.
Moved maintenance instructions from Appendix A Decontamination and Maintenance into a
new Chapter 6, Preventive Maintenance. Renamed Appendix A to Decontamination.
In the new chapter 6, added a recommended Maintenance Schedule. Streamlined the
sequence of maintenance steps to better represent actual practice. Removed the limitation
that removing/cleaning optical probes only be performed for models with the external
dispenser module.
Updated the Error Code listing in Appendix B with new information.
BioTek Instruments, Inc.
Revision History |
xi
Rev
Date
Changes
Additional cosmetic improvements.
I
6/06
Preface: Added Directive 98/79/EC: In Vitro Diagnostics (if labeled for this use) and Directive
2002/96/EC: Waste Electrical and Electronic Equipment (page xii). Added “Consult
instructions for use” and “In vitro diagnostic medical device” safety symbols (page xiv).
Expanded Intended Use Statement (page xv).
®
Instrument Qualification: Under “Required Materials” removed the Corning Costar #3590
plate as a recommendation for the Corners Test. Plate type recommendations are the same
for both Corners & Sensitivity tests. Under “Titration Dyes” step C, changed PBS volume from
7.800 ml to the more appropriate 6.800 ml.
J
7/11
General: Removed references to outdated software KC4 and KCjunior. Updated Gen5
instructions for Gen5 2.x. Added information about new USB port and driver software.
Preface: Updated regulatory and hazard information to match current standards. Updated
TAC contact information.
Chapter 1: Added Package Contents and Optional Accessories sections.
Chapter 3: Removed serial and parallel pin diagrams. Added Install Software/Connect to
Computer section.
Chapter 4: Added Getting Started with Gen5 section
Chapter 5: Streamlined the Liquid Test procedures. Added support for BioTek fluorescence
liquid test kits.
Appendix B: Added probable causes and solutions to Error Codes 0200 and 0601.
Removed former Appendix C: Computer Control
ELx808 Operator’s Manual
xii | Preface
Intended Use Statement
•
The FLx800 is a multi-detection microplate reader. The performance characteristics
of the data reduction software have not been established with any laboratory
diagnostic assay. The user must evaluate this instrument and (if used) PC-based
software in conjunction with their specific assay(s). This evaluation must include
the confirmation that performance characteristics for the specific assay(s) are met.
•
This product may be used for In Vitro Diagnostic, research and development, or
other non-clinical purposes.
Quality Control
It is considered good laboratory practice to run laboratory samples according to
instructions and specific recommendations included in the package insert for the test to be
conducted. Failure to conduct Quality Control checks could result in erroneous test data.
Warranty and Product Registration
Take a moment to review the Warranty information that shipped with your product.
Please also register your product with BioTek to ensure that you receive important
information and updates about the product(s) you have purchased. You can register online
through the Customer Resource Center at www.biotek.com or by calling 888/451-5171 or
802/655-4740.
Repackaging and Shipping

If you need to ship the instrument to BioTek for service or repair,
contact BioTek for a Return Materials Authorization (RMA)
number, and be sure to use the original packing materials. Other
forms of commercially available packaging are not recommended
and can void the warranty. If the original packing materials have
been damaged or lost, contact BioTek for replacement packing.
BioTek Instruments, Inc.
Warnings |
xiii
Warnings
Operate the instrument on a flat surface away from excessive humidity.
Bright sunlight or strong incandescent light can reduce the linear performance
range of the instrument.
Measurement values may be affected by extraneous particles (such as dust) in the
microplate wells. A clean work area is necessary to ensure accurate readings.
When operated in a safe environment according to the instructions in this
document, there are no known hazards associated with the instrument. However,
the operator should be aware of certain situations that could result in serious
injury; these vary depending on the instrument type. See Hazards and
Precautions.
Hazards
The following hazard warnings are provided to help avoid injury:
Warning! Power Rating. The instrument’s power supply or power cord must
be connected to a power receptacle that provides voltage and current within the
specified rating for the system. Use of an incompatible power receptacle may
produce electrical shock and fire hazards.
Warning! Electrical Grounding. Never use a plug adapter to connect primary
power to the external power supply. Use of an adapter disconnects the utility
ground, creating a severe shock hazard. Always connect the power cord directly
to an appropriate receptacle with a functional ground.
Warning! Internal Voltage. Always turn off the power switch and unplug the
power supply before cleaning the outer surface of the instrument or removing its
top case.
Warning! Lubricants. Do not apply lubricants to the microplate carrier or
carrier track. Lubricant on the carrier mechanism or components in the carrier
compartment will attract dust and other particles, which may obstruct the carrier
path and cause the instrument to produce an error.
Warning! Liquids. Avoid spilling liquids on the instrument; fluid seepage into
internal components creates a potential for shock hazard or instrument damage.
If a spill occurs while a program is running, abort the program and turn the
instrument off. Wipe up all spills immediately. Do not operate the instrument if
internal components have been exposed to fluid.
ELx808 Operator’s Manual
xiv | Preface
Warning! Potential Biohazards. Some assays or specimens may pose a
biohazard. Adequate safety precautions should be taken as outlined in the assay’s
package insert. This hazard is noted by the symbol shown here. Always wear
safety glasses and appropriate protective equipment, such as chemically resistant
rubber gloves and apron.
Warning! Unspecified Use. Failure to operate this equipment according to the
guidelines and safeguards specified in this manual could result in a hazardous
condition.
Warning! Hot Surface. The lamp assembly is hot when the instrument is
turned on. Turn off the reader and allow the lamp to cool down before
attempting to replace it.
Warning! Pinch Hazard. Some areas of the instrument or its components can
present pinch hazards when the instrument is operating. These areas are marked
with the symbol shown here. Keep hands/fingers clear of these areas when the
instrument is operating.
Warning! Software Quality Control. The operator must follow the
manufacturer’s assay package insert when modifying software parameters and
establishing reading, washing, or dispensing methods. Failure to conduct
quality control checks could result in erroneous test data.
Warning! Reader Data Reduction Protocol. The onboard assay software
will flag properly defined controls when they are out of range. The software will
present the data with the appropriate error flags for the operator to determine
control and assay validity.
Precautions
The following precautions are provided to help avoid damage to the instrument:
Caution: Service. The instrument should be serviced by BioTek authorized
service personnel. Only qualified technical personnel should perform
troubleshooting and service procedures on internal components.
Caution: Environmental Conditions. Do not expose the instrument to
temperature extremes. For proper operation, ambient temperatures should remain
within the range listed in the Specifications section. Performance may be
adversely affected if temperatures fluctuate above or below this range. Storage
temperature limits are broader.
Caution: Sodium Hypochlorite. Do not expose any part of the instrument to the
recommended diluted sodium hypochlorite solution (bleach) for more than 20
minutes. Prolonged contact may damage the instrument surfaces. Be certain to
rinse and thoroughly wipe all surfaces.
BioTek Instruments, Inc.
Precautions |
xv
Caution: Chemical Compatibility. Some chemicals may cause irreparable
damage to the instrument. The following chemicals have been deemed safe for use
in the instrument: buffer solutions (such as PBS), saline, surfactants, deionized
water, 70% ethyl, isopropyl, or methyl alcohol, 40% formaldehyde, and 20%
sodium hydroxide. Never use acetic acid, DMSO, or other organic solvents. These
chemicals may cause severe damage to the instrument. Contact BioTek for more
information and prior to using other questionable chemicals.
Caution: Power Supply. Only use the power supply shipped with the
instrument. Operate this power supply within the range of line voltages listed on
it.
Caution: Disposal. This instrument contains printed circuit boards and wiring
with lead solder. Dispose of the instrument according to Directive 2002/96/EC,
“on waste electrical and electronic equipment (WEEE),” or local ordinances.
Caution: Warranty. Failure to follow preventive maintenance protocols may
void the warranty.
Caution: Shipping Hardware. All shipping hardware must be removed before
operating the instrument and reinstalled before repackaging the instrument for
shipment.
Caution: Electromagnetic Environment. Per IEC 61326-2-6 it is the user’s
responsibility to ensure that a compatible electromagnetic environment for this
instrument is provided and maintained in order that the device will perform as
intended.
Caution: Electromagnetic Compatibility. Do not use this device in close
proximity to sources of strong electromagnetic radiation (e.g., unshielded
intentional RF sources), because these may interfere with the proper operation.
ELx808 Operator’s Manual
xvi | Preface
CE Mark
Based on the testing described below and information contained
herein, this instrument bears the CE mark
 See the Declaration of Conformity for more information.
Directive 2004/108/EC: Electromagnetic Compatibility
Emissions—Class A
The system has been type-tested by an independent, accredited testing laboratory
and found to meet the requirements of EN 61326-1 and EN 61326-2-6: Class A for
Radiated Emissions and Line Conducted Emissions.
Verification of compliance was conducted to the limits and methods of EN 55011 –
(CISPR 11) Class A. In a domestic environment it may cause radio interference, in
which case you may need to mitigate the interference.
Immunity
The system has been type-tested by an independent, accredited testing laboratory
and found to meet the requirements of EN 61326-1 and EN 61326-2-6 for Immunity.
Verification of compliance was conducted to the limits and methods of the
following:
EN 61000-4-2, Electrostatic Discharge
EN 61000-4-3, Radiated EM Fields
EN 61000-4-4, Electrical Fast Transient/Burst
EN 61000-4-5, Surge Immunity
EN 61000-4-6, Conducted Disturbances from RFI
EN 61000-4-11, Voltage Dips, Short Interruptions and Variations
Directive 73/23/EEC Low Voltage (Safety)
The system has been type-tested by an independent testing laboratory and was
found to meet the requirements of EC Directive 73/23/EEC for Low Voltage.
Verification of compliance was conducted to the limits and methods of the
following:
EN 61010-1 (2001) 2nd Edition. “Safety requirement for electrical equipment for
measurement, control and laboratory use. Part 1, General requirements.”
BioTek Instruments, Inc.
Electromagnetic Interface and Susceptibility |
xvii
Directive 2002/96/EC: Waste Electrical and Electronic Equipment
Disposal Notice: This instrument contains printed circuit boards and wiring with
lead solder. Dispose of the instrument according to Directive 2002/96/EC, “on waste
electrical and electronic equipment (WEEE)” or local ordinances.
Directive 98/79/EC: In Vitro Diagnostics
•
Product registration with competent authorities.
•
Traceability to the U.S. National Institute of Standards and Technology (NIST).
Electromagnetic Interface and Susceptibility
USA FCC CLASS A
Warning: Changes or modifications to this unit not expressly approved by the
manufacturer could void the user's authority to operate the equipment.
This equipment has been tested and found to comply with the limits for a Class A
digital device, pursuant to Part 15 of the FCC Rules.
These limits are designed to provide reasonable protection against harmful
interference when the equipment is operated in a commercial environment. Like all
similar equipment, this equipment generates, uses, and can radiate radio frequency
energy and, if not installed and used in accordance with the instruction manual,
may cause harmful interference to radio communications. Operation of this
equipment in a residential area is likely to cause interference, in which case the
user will be required to correct the interference at their own expense.
Canadian Department of Communications Class A
This digital apparatus does not exceed Class A limits for radio emissions from
digital apparatus set out in the Radio Interference Regulations of the Canadian
Department of Communications.
Le present appareil numerique n'émet pas de bruits radioelectriques depassant les
limites applicables aux appareils numerique de la Class A prescrites dans le
Reglement sur le brouillage radioelectrique edicte par le ministere des
Communications du Canada.
ELx808 Operator’s Manual
xviii | Preface
Safety Symbols
Some of these symbols appear on the instrument or accessories:
Alternating current
Courant alternatif
Wechselstrom
Corriente alterna
Corrente alternata
Both direct and alternating current
Courant continu et courant alternatif
Gleich - und Wechselstrom
Corriente continua y corriente alterna
Corrente continua e corrente alternata
Direct current
Courant continu
Gleichstrom
Corriente continua
Corrente continua
Earth ground terminal
Borne de terre
Erde (Betriebserde)
Borne de tierra
Terra (di funzionamento)
On (Supply)
Marche (alimentation)
Ein (Verbindung mit dem
Netz)
Conectado
Chiuso
Protective conductor terminal
Borne de terre de protection
Schutzleiteranschluss
Borne de tierra de protección
Terra di protezione
Off (Supply)
Arrêt (alimentation)
Aus (Trennung vom Netz)
Desconectado
Aperto (sconnessione dalla
rete di alimentazione)
Caution (refer to accompanying
documents)
Attention (voir documents
d’accompanement)
Achtung siehe Begleitpapiere
Atención (vease los documentos
incluidos)
Attenzione, consultare la doc annessa
Warning, risk of electric shock
Attention, risque de choc
électrique
Gefährliche elektrische schlag
Precaución, riesgo de
sacudida eléctrica
Attenzione, rischio di scossa
elettrica
Warning, risk of crushing or pinching
Attention, risque d’écrasement et
pincement
Warnen, Gefahr des Zerquetschens und
Klemmen
Precaución, riesgo del machacamiento y
sejeción
Attenzione, rischio di schiacciare ed
intrappolarsi
Warning, hot surface
Attention, surface chaude
Warnen, heiße Oberfläche
Precaución, superficie caliente
Attenzione, superficie calda
Warning, potential biohazards
Attention, risques biologiques potentiels
Warnung! Moegliche biologische
Giftstoffe
Atención, riesgos biológicos
Attenzione, rischio biologico
BioTek Instruments, Inc.
Safety Symbols |
In vitro diagnostic medical
device
Dispositif médical de
diagnostic in vitro
Medizinisches In-VitroDiagnostikum
Dispositivo médico de
diagnóstico in vitro
Dispositivo medico
diagnostico in vitro
Consult instructions for use
Consulter la notice d’emploi
Gebrauchsanweisung beachten
Consultar las instrucciones de
uso
Consultare le istruzioni per
uso
ELx808 Operator’s Manual
xix
Separate collection for electrical and
electronic equipment
Les équipements électriques et
électroniques font l’objet d’une collecte
sélective
Getrennte Sammlung von Elektro- und
Elektronikgeräten
Recogida selectiva de aparatos eléctricos y
electrónicos
Raccolta separata delle apparecchiature
elettriche ed elettroniche
xx | Preface
BioTek Instruments, Inc.
Chapter 1
Introduction
This chapter provides an introduction to hardware and software
features and technical specifications of the FLx800. See Chapter 2,
Instrument Description for more in-depth information.
Product Overview................................................................ 2
Hardware Features .............................................................. 3
Software Features ............................................................... 4
Package Contents ............................................................... 5
Optional Accessories ........................................................... 5
Technical Specifications ....................................................... 6
2 | Chapter 1: Introduction
Product Overview
BioTek’s FLx800 is a fluorescence and luminescence measurement system that can operate
as a stand-alone instrument, or serve as part of a larger laboratory network.
The FLx800 performs measurements using a photomultiplier tube (PMT), which is housed
inside a light-impermeable detection compartment. It is a single-channel instrument with
stationary optics. Depending on the model, the instrument may be equipped with a
bottom probe, a top probe, or both.
The plate carrier moves in the X and Y axes, and supports 6-, 12-, 24-, 48-, 96-, and 384-well
standard microplates, 96-well Hellma microplates, and 96-well Metric microplates (see
Technical Specifications on page 6 for more information). The excitation and emission
filter wheels are removable for filter exchange and cleaning.
The on-board software can store up to 75 assays, and supports the endpoint read method.
Definable assay parameters include excitation and emission filters, probe selection, plate
geometry, samples per well, delay before sampling, delay between samples, sensitivity,
and numerous plate mapping and data reduction options.
The instrument’s onboard processor, 2 x 24 character LCD, and membrane keypad allow
easy definition and management of assay protocols, templates, formulas and data. Results
can be output in a printed report format, or exported for use in a variety of microplatebased data manipulation applications.
In addition, BioTek’s Gen5 software package can be used to control the FLx800 and
perform data reduction, print customized reports, and more. The kinetic read method and
reagent dispense and read method are supported through computer control. Optional
incubation and shake functions are available and controlled via BioTek’s Gen5
software.
Also available on the FLx800 is optional reagent dispensing with a single injector that
supports top or bottom fluorescence/luminescence measurements. The injector has two
manually configurable positions so that dispensing can occur in the well directly below the
top probe, or in the well directly above the bottom probe. Reagent dispensing is only
available for incubation instruments.
BioTek Instruments, Inc.
Hardware Features |
3
The FLx800 is available in the following models:
FLx800T:
Top probe (3 mm) only, low-noise PMT
FLx800TB:
Top and bottom probes, low-noise PMT
FLx800TBI:
Top and bottom probes, incubation to 50ºC and shaking, low-noise
PMT
FLx800TBP:
Top and bottom probes, top/bottom PCR tube and low-noise PMT
FLx800TBID:
Top and bottom probes, low-noise PMT, incubation to 50ºC and
shaking; includes dispenser module
FLx800TE:
Top probe (3 mm) only, red extended PMT
FLx800TBE:
Top and bottom probes, red extended PMT
FLx800TBIE:
Top and bottom probes, incubation to 50ºC and shaking, red
extended PMT
FLx800TBEP:
Top and bottom probes, top/bottom PCR tube, and red extended
PMT
The TBP and TBEP models are specially designed to accommodate
plates and PCR tubes with heights up to 1.250".
To effectively read 96-well microplates using the top probe, a plate
carrier adapter is required (BioTek part number 7080522) to raise the
microplate and bring it closer to the probe. Microplates with heights
up to 0.600" can be read using this adapter.
FLx800TBIDE:
Top and bottom probes, incubation to 50ºC and shaking, red
extended PMT; includes dispenser module
Hardware Features
The FLx800 is equipped with the following:
•
Single-channel fluorometer
•
X-Y carrier movement
•
Low-noise photomultiplier (PMT) with the FLx800T, TB, TBI, TBID, and TBP
models
•
Red extended photomultiplier (PMT) with the FLx800TBE, TBEP, TBIE, TBIDE, and
TE models
•
Excitation and Emission filter wheels, each with 4 filter positions
•
Stationary optics
 Top and/or bottom probe (depending on model)
 Top probe height is user-adjustable
•
2-line x 24-character Liquid Crystal Display (LCD)
FLx800 Operator’s Manual
4 | Chapter 1: Introduction
•
Membrane keypad with 25 alphanumeric keys
•
Capability of reading 6-, 12-, 24-, 48-, 96-, and 384-well standard microplates, 96well Hellma microplates, and 96-well Metric microplates (see Technical
Specifications on page 6 for more information)
•
External 24-volt power supply which runs on 100-240 VAC ± 10% @ 50-60 Hz
•
Optional incubated read chamber with shaking functionality
•
One serial communications port
•
One parallel port
•
One USB port (some models)
•
Optional reagent dispensing
 Manually adjustable single injector supporting top or bottom
fluorescence/luminescence measurements
 Capability of dispensing into the well directly below the top probe or into the
well directly above the bottom probe
 Reagent dead volume less than 1.0 ml with dead volume recovery
 Dispense volume range of 5-1000 µl with a programmable 5-20 µl tip prime
 Dispenser priming volume range of 5-5000 µl
 Well mode and plate mode kinetics supported
 Capability of dispensing to 6-, 12-, 24-, 48-, and 96-well plates.
Software Features
The on-board, menu-driven software supports:
•
Fluorescence and luminescence assays
•
Endpoint reading method
•
1, 2, or 3 filtersets per assay
•
For each filterset, the selection of:
 Excitation filter, emission filter
 Probe selection (top or bottom), if the instrument is equipped with two probes
 Number of samples per well
 Delay before sampling, delay between samples
 Sensitivity value or automatic sensitivity selection
•
The option to automatically subtract the results of Filterset 2 from the results of
Filterset 1
BioTek Instruments, Inc.
Package Contents |
5
•
Curve-fitting methods: 4-parameter, cubic, quadratic, linear, 2-P, cubic-spline and
point-to-point
•
Transformation and formula calculations for more complex mathematical
operations, including blank, control, and assay validation
•
Data reduction for all plate types listed under Hardware Features
•
Storage of up to 75 assays
•
Storage of up to 6 sets of results at a time
•
Reader diagnostics and software version information
Package Contents
 Package contents and part numbers are subject to change. Please
contact BioTek Customer Care with questions.
•
FLx800 Multi-Detection Microplate Reader
•
Power cord (PN varies according to country of use), Power Supply (PN 61062)
•
Operator’s Manual (PN 7081000)
•
Dust Cover
•
Serial Cable (PN 75053 9-25p or 75034 9-9p, depending on the model)
•
If applicable, USB Cable (PN 75108) with USB Driver Software (PN 7090204)
Optional Accessories
 Accessory availability and part numbers are subject to change. Contact
BioTek Customer Care with questions, or visit www.biotek.com and
use the Accessories search tool.
•
Replacement lamp assembly (PN 7080500 Cable Asby Bulb)
•
FLx800 IQ/OQ/PQ Package (PN 7080532)
•
Gen5 software (visit BioTek.com or contact your local dealer for details)
•
Adapter to connect the reader to a USB-only printer (PN 75135)
•
Fluorescence test plate (PN 7092092)
•
Plate carrier adaptor (PN 7080522)
•
For the Fluorescence Liquid Tests
 Sodium Fluorescein powder (1 mg vial) (PN 98155)
 Liquid Test Kit using Sodium Fluorescein (PN 71600013)
 Liquid Test Kit using Methylumbelliferone (MUB) (PN 7160012)
FLx800 Operator’s Manual
6 | Chapter 1: Introduction
Technical Specifications
Microplates
Accommodates these
standard-size
microplates:
6-well (2 x 3)
96-well (8x12)
12-well (3 x 4)
384-well (16 x 24)*
24-well (4 x 6)
96-well Hellma (8 x 12)
48-well (6 x 8)
96-well Metric (8 x 12, with
9 mm well-to-well spacing)
*384-well plates are not supported for the dispense/read
feature.
Maximum plate height:
0.800” (20.32 mm) for all models except
1.250” (31.75 mm) for the FLx800 TBEP and TBP models
Power
The instrument is operated from a 24-volt external power supply that is compatible
with 100-240 V~; 50-60 Hz. Wattage applied is based on the instrument
configuration.
Sensitivity
Specifications apply to regular mode of reading
Bottom reading,
5 mm optical probe
10 pg/mL solution of Sodium Fluoroscein in PBS
150 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 528/20
Hellma 96-well quartz plate
62.5 ng/mL solution of Propidium Iodide in PBS
150 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 645/40
Corning Costar 96-well plate with black sides and
a clear bottom
Top reading,
3 mm optical probe
20 pg/Ml solution of Sodium Fluoroscein in PBS
150 µL per well signal-to-noise ratio greater than 2
Excitation 485/20, Emission 528/20
Hellma 96-well quartz plate
0.16 ng/ml solution of Methylumbelliferone in CBB
300 µL per well signal-to-noise ratio greater than 2
Excitation 360/40, Emission 460/40
Corning Costar black strips
BioTek Instruments, Inc.
Technical Specifications |
Optical
Specifications apply to flat- and round-bottom full well plates
Lamp Type:
Tungsten halogen 20W power, 12V (user-changeable)
Lamp Life:
1000 hours
Filter Wheels:
Excitation and Emission, each with four filter positions
Excitation Range:
300 to 650 nm
Emission Range:
340 to 700 nm, or 340 to 800 nm for extended-range
models
Detector:
Photomultiplier tube (PMT)
Low-noise PMT for standard models
Red extended PMT for extended-range models
Probe Diameters:
Fully randomized quartz fiber bundles
5.0 mm general-purpose probe (bottom probe only)
3.0 mm general-purpose probe (top probe only)
Probe Selector:
Selection of top or bottom read position through the
on-board or PC software, for models equipped with both
top and bottom probes (TBxx)
Incubation
Temperature Range:
“I” models incubate from 4°C over ambient to 50°C
(or to 65°C for FLx800TI)
Temperature Variation:
± 0.75°C well variation with a covered plate at 37°C
Environmental
Operating Conditions:
15°C to 35°C during operation
Relative Humidity:
10 to 85%, non-condensing
Physical
Dimensions:
16.0" D x 15.5" W x 9.0" H
40.6 cm D x 14.0 cm W x 22.9 cm H
Weight:
30 lbs. / 13.6 kg
FLx800 Operator’s Manual
7
8 | Chapter 1: Introduction
Dispense/Read
Specifications apply to models with the dispense module
Well mode, kinetic
specifications:
Supports single or dual filter set measurements. Single
sensitivity for all readings. Dual filters must be positioned
next to each other on the filter wheel.
Single programmable dispense event for which time of
dispense relative to samples is indicated. During
dispense, readings are inhibited.
Up to two programmable kinetic sampling windows
Starting lag time (0-10 minutes)
Number of samples (1-300)
Sample Interval:
Single filter set (20 ms-12 sec)
Two filter sets w/single filter switch (140 ms-12 sec)
Two filter sets w/two filter switches (240 ms-12 sec)
For light shuttering, add 100 ms to minimum
sampling interval for 2 sets, 1 switch; add 200 ms
for 2 sets, 2 switches
Light shutter blocks light when not measuring to inhibit
photo bleaching. During light shuttering, two blocking
filters must be installed in the Excitation Filter wheel.
Plate mode, kinetic
specifications:
Supports single and dual filter set measurements. Single
sensitivity for all readings. Dual filters must be positioned
next to each other.
Single programmable dispense event that can be set to
execute on any defined kinetic read. When a dispense
action is set to execute on a read then the dispense will
occur first then the read.
Number of Kinetic Reads (1-300)
Kinetic Interval (1-9999, 1 sec granularity)
Light shutter blocks light when not measuring to inhibit
photo bleaching. During light shuttering, two blocking
filters must be in the Excitation Filter wheel.
Volume Range:
5-1000 µL
Delay Range:
0-6000 seconds
Accuracy:
± 1 µL or 2%, whichever is greater
Precision:
< 2% for 50-200 µL
< 4% for 25-49 µL
< 7% for 10-24 µL
< 10% for 5-9 µL
BioTek Instruments, Inc.
Chapter 2
Instrument Description
This chapter discusses the principles of operation and introduces
important external and internal components. The information provided
here is important for the operation, customization, and maintenance of
the instrument.
Principles of Operation ....................................................... 10
External Components ........................................................ 11
External Components for the Dispenser Module .................... 13
Internal Components for Models with Dispense Capability ...... 15
Excitation and Emission Filter Wheels .................................. 17
Filter Position Requirements for Dispense / Read Assays ... 19
Configuration for Luminescence Measurements ................ 19
Top Optical Probe Adjustment ............................................ 20
Lamp Assembly ................................................................ 22
10 | Chapter 2: Instrument Description
Principles of Operation
The FLx800 reader is a microprocessor-driven instrument with sufficient memory for
performing all functions described in Chapter 4, Operation, and for the storage of up to 75
assays. In stand-alone mode, assays are defined and run via the instrument’s keypad.
Alternatively, the FLx800 can be controlled by an external PC.
When a plate read is initiated, the plate carrier is drawn into the measurement chamber. The
plate carrier moves in the X and Y axes to align each microwell with the top or bottom probe,
as specified in the assay. The assay defines additional reading parameters such as plate
geometry, excitation and emission filters, number of measurements per well, delay before
sampling, delay between samples, and the sensitivity setting. In addition, for dispense/read
methods, the assay defines the dispense volume, dispense rate, and the delay after dispense.
Measurements are performed using a photomultiplier tube (PMT), housed inside a lightimpermeable compartment. The microprocessor converts each well’s set of measurements into
a Relative Fluorescence Unit (or Relative Luminescence Unit). When all measurements have
been taken for every well on the plate, data reduction is performed according to the defined
assay parameters, which can include transformation formulas and/or curve fitting. When data
reduction is complete, the data is ready for output via the serial port to an external computer,
or via the parallel port to a printer.
Incubated models employ a three-zone heater using a thermistor feedback. A specific
incubation carrier position is used during wait intervals between consecutive reads. The door,
when open, will disable the heating plate that is located on the door assembly. System selfchecks verify that all Incubation Zones are functioning correctly.
All incubated instruments have shake capability.
Readers equipped with a reagent injector have an external syringe drive that delivers reagent
to the assay wells. A 250-µl syringe and microstepping drive deliver accurate volumes of 5-200
µl per stroke. Opaque tubing and a cover over the syringe drive prevent stray light from
entering the reader.
Figure 1 shows the FLx800 external components.
Figure 3 and Figure 4 show the FLx800 external components for the Dispenser Module.
Figure 5 shows the FLx800 internal components for the Dispenser Module.
BioTek Instruments, Inc.
External Components |
External Components
Access cover to raise/
lower top optical probe
Microplate
carrier
Lamp assembly
and Excitation
filter wheel
compartments
Power switch
Front panel/keypad
Emission filter wheel
Tubing port
(models with
Dispense capability)
Power inlet
Parallel port
BACK PANEL
Serial port
USB port
Dispenser module
port (models with
Dispense capability)
Shipping bracket in
its storage location
Figure 1: External components of the FLx800 (some models are
equipped with a 25-pin serial port and no USB)
FLx800 Operator’s Manual
11
12 | Chapter 2: Instrument Description
•
The power switch is located on the right side of the instrument.
•
The front panel features a 2 x 24-character Liquid Crystal Display (LCD), and a 25key keypad. Through this interface you can create and modify fluorescence and
luminescence assays, read plates, store and print results, send results to an external
computer, and more. See Chapter 3, Operation for more information.
•
The microplate carrier supports the microplates and adapter plates described in
Chapter 1, under Technical Specifications. A spring clip holds the plate securely in
place. The microplate carrier access door with its magnetic closure helps to ensure a
light-impermeable measurement chamber.
•
When a plate read is initiated, the plate carrier is drawn into the measurement
chamber, and then moves in the X and Y axes to align each microwell with the top or
bottom probe, as specified in the assay. When the read is complete, the plate carrier is
returned to its full-out position.
•
The back panel contains the power inlet, communication port(s), parallel port,
dispenser port, and tubing port (dispense/read instruments only). See Setting Up
the FLx800 in Chapter 3 for more information.
•
Opening the hinged door on the front of the instrument accesses the lamp assembly
and Excitation and Emission filter wheels. The door is held in place with two magnets.
•
A diagram showing the location of the lamp assembly and the orientation of the
Excitation and Emission filter wheels is printed on the inside of the hinged door.
•
The four screws on the top of the instrument hold the top optical probe access
cover in place. For FLx800 readers equipped with a top optical probe, this cover must
be removed to adjust the height of the probe. See Top Optical Probe Adjustment on
page 20 for more information.
 Figure 2 shows the top optical probe access cover for models FLx800T, B,
TB, TBI, and TBE. The access cover for the FLx800TBEP model is shaped
differently, as shown below:
Figure 2: Top optical probe access cover for the FLx800TBEP
BioTek Instruments, Inc.
External Components for the Dispenser Module |
External Components for the Dispenser Module
Dispenser
module,
front view
Storage bag for the
stylus and priming
trough, user-applied
to the dispenser
module’s back panel
Tubing port
Dispenser
module cable,
connected to
ports on the
side of the
dispenser
module and the
back panel of
the FLx800
Figure 3: Orientation of the FLx800 Dispenser Module (front and rear)
FLx800 Operator’s Manual
13
14 | Chapter 2: Instrument Description
Outlet tube
17” (46 cm)
Three-way valve
Inlet tube
8” (20 cm)
Syringe
Thumbscrew
Supply bottle,
in holder
Figure 4: External components for the FLx800 Dispenser Module
•
The dispenser module cable sends power and commands from the reader to the
syringe drive and returns sensor output from the syringe drive to the reader.
•
The two dispenser module ports are mounted in the left side of the dispenser
module and in the base of the back panel of the reader. These mate with the dispenser
module cable.
•
The tubing port (reader back panel) is a threaded port that mates with the fitting
from the outlet tube of the syringe drive. One end of the outlet tube must be screwed
into the port. Use fingers only (no tools) to tighten the fitting.
•
A 250-µl syringe is used to transport fluid from a reagent vessel to the assay well. A
thumbscrew is used to connect the bottom of the syringe to a metal bracket on the
dispenser module. The syringe is serviceable by the user.
•
The inlet tube is a short piece of opaque PTFE (Teflon) tubing connected to a stainless
steel straw on one end and a threaded fitting on the other that is used to aspirate fluid
from the supply vessel into the syringe. The threaded fitting connects to the left side of
the valve and the steel straw goes into the supply bottle.
•
The outlet tube is an opaque PTFE tube with threaded fittings on each end that is
used to deliver fluid from the syringe to the reader. It connects to the right side of the
valve above the syringe and to the tubing port at the rear of the reader.
•
The three-way valve is used to switch the syringe flow from the inlet tube to the
outlet tube.
BioTek Instruments, Inc.
Internal Components for Models with Dispense Capability |
Internal Components for Models with Dispense
Capability
Tip
priming
trough
Priming
plate
Thumbscrew
Thumbscrew
Injector head
Figure 5: Internal components for the FLx800 Dispenser Module
FLx800 Operator’s Manual
15
16 | Chapter 2: Instrument Description
•
The injector head mates the internal tube to a dispense probe and mounts on the
optic probe block. The injector head is positioned to dispense into a well above the
bottom probe.
•
The internal tube is an opaque PTFE (Teflon) tube, with threaded fittings on each
end, which is used to deliver fluid from the tube port at the rear of the reader to the
injector head.
•
The tip priming trough is a small, removable priming cup located in the right rear of
the carrier, used for performing the Tip Prime. The purpose of Tip Prime is to
compensate for any fluid loss at the dispense tip due to evaporation since the last
dispense. The prime cup holds up to 2 ml of liquid and must be periodically emptied
and cleaned by the user.
•
The prime operation dispenses fluid into a priming plate (BioTek PN 7192135), which
must be placed in the carrier prior to priming.
Priming plate
Tip priming trough
Figure 6: Priming plate and tip priming trough
BioTek Instruments, Inc.
Excitation and Emission Filter Wheels |
17
Excitation and Emission Filter Wheels
All FLx800 models are equipped with one Excitation filter wheel and one Emission filter wheel
(see Figure 7 and Figure 8 below). The Excitation filter selects the narrow band of light to
which the sample will be exposed. The Emission filter selects the band of light with the
maximum fluorescence signal, to be measured by the photo-multiplier (PMT).
Each filter wheel can contain up to four filters and/or black “plugs.” Each filter and plug is
held securely in place with a C-clip filter retainer. Note: Each filter has an arrow printed on its
side to indicate the proper direction of light through the filter.
Refer to page 19 for filter position requirements for Dispense/Read assays.
Direction
of light
Supporting
metal bracket
Filter wheel
485/20
Thumbscrew
Figure 7: Side view of EXCITATION filter wheel, showing proper filter orientation.
Direction
of light
528/20
Note the difference in filter orientation between
the Emission and the Excitation filter wheels
Figure 8: Side view of EMISSION filter wheel, showing proper filter orientation.
FLx800 Operator’s Manual
18 | Chapter 2: Instrument Description

The filter wheels can be removed if their contents need to be
changed.
Notes: (1) the Excitation and Emission filter wheels are not
interchangeable, (2) filter direction within a filter wheel is
important, and the direction differs depending on the filter
wheel, (3) each filter is marked with an arrow indicating the
proper direction of light, and (4) if a filter wheel’s contents are
changed, the FLx800’s internal filter tables must be updated
accordingly.
Refer to Figure 7 and Figure 8 on the previous page for proper
filter orientation. See also Excitation and Emission Filter
Tables in Chapter 2.
To remove a filter wheel and change its contents:
1.
Important! Turn off the instrument.
2.
Pull down the hinged door on the front of the instrument. Observe the two
thumbscrews within the compartment. The left thumbscrew holds the Excitation filter
wheel in place; the right secures the Emission filter wheel. The Emission filter wheel
will “spring” out when removed. This is because a shutter behind the wheel closes
quickly to protect the PMT.
3.
Remove the thumbscrew, then slide the filter wheel’s supporting metal bracket straight
out of the compartment.
4.
To remove a filter or plug:
•
Turn the filter wheel to align the desired filter with the hole in the supporting
bracket.
•
Place the bracket on a flat surface, with the filter wheel facing down.
•
Prepare a multi-layered “cushion” of lens paper. Using your finger covered with
the lens paper, gently push against the filter until it and its C-clip retainer pop out.

Important! When removing or replacing a filter or C-
clip filter retainer, do not use a sharp instrument! Use
several layers of lens paper and your finger to remove and
replace filters and clips. Using a sharp instrument, such as
a flat screwdriver, will scratch the filter surface and render
the filter unusable.
BioTek Instruments, Inc.
Excitation and Emission Filter Wheels |
5.
6.
19
To replace a filter or plug:
•
Hold the metal bracket with the filter wheel facing up.
•
Properly orient the filter or plug, then drop it into the desired filter wheel
•
Using your fingers, squeeze the sides of the C-clip filter retainer, then insert it into
the top of the hole containing the new filter. Cover your finger with several layers
of lens paper, then push down on all sides of the C-clip until it sits flush against the
filter.
•
Clean both sides of the filter with lens paper.
location.
To replace a filter wheel:
•
Ensure that all filters and/or plugs are inserted properly (see step 5).
•
Slide the filter wheel back into its chamber.
•
Replace the thumbscrew.
Filter Position Requirements for Dispense / Read Assays
1.
Single filter set assays:
If light shuttering is specified, a blocking filter (Plug) must be placed in the excitation
filter wheel in one of the two positions next to the excitation filter that is specified in
the assay protocol.
2.
Dual (ratioed) filter set assays:
•
The two excitation filters specified in the assay protocol must be positioned next to
each other in the excitation filter wheel.
•
The two emission filters specified in the assay protocol must be positioned next to
each other in the emission filter wheel.
•
If light shuttering is specified, two blocking filters (Plugs) must be placed in the
excitation filter wheel.
Configuration for Luminescence Measurements
For best results when taking luminescence measurements, the Excitation filter wheel
should have no empty locations, and it should have at least one “plug” (also referred to as
a “dummy filter”) installed, to prevent light from reaching the samples.
If your tests require that the light emitted from the samples remain unfiltered, the
Emission filter wheel should have an empty location in it.

Important! If you change the contents of a filter wheel, you
must update the FLx800’s filter table. The FLx800 does not
automatically detect which filters are installed. See
Configuring the Excitation and Emission Filter Tables in
Chapter 3.
FLx800 Operator’s Manual
20 | Chapter 2: Instrument Description
Top Optical Probe Adjustment
FLx800 “T” models are equipped with a top optical probe, positioned above the plate
carrier in the measurement chamber.
The top probe mechanism has a manual vertical adjustment to accommodate microplates
with heights up to 0.800" (1.250" for FLx800TBEP model). The height of top probe can be
adjusted and tested with a particular microplate prior to actual plate reading (see next page).
To access the top probe mechanism, remove the four screws from the top of the instrument
and lift off the cover, as shown in Figure 9. A close-up of the top probe is shown in Figure
10.

Important! Turn the instrument off before removing the
cover to access the top optical probe mechanism.
Figure 9: Accessing the top probe mechanism to adjust the height.
The inset shows the probe access cover for the FLx800TBEP model.
BioTek Instruments, Inc.
Top Optical Probe Adjustment |
As the adjustment screw is
turned, this block (with the
optic probe cable attached)
moves up or down
21
Probe height adjustment,
turn counter-clockwise
to lower the optic probe
Stationary block
Optic probe cable
Figure 10: Close-up of the top probe mechanism.
The top probe mechanism consists of a stationary block with a screw adjustment, connected to
a movable block that slides in the vertical axis as the screw is turned. The optic probe cable is
affixed to the movable block. As the block slides down, the optic probe moves closer toward
the microplate.
To adjust the height of the top probe to accommodate a specific microplate:
1.
Important! Turn off the instrument.
2.
Remove the four thumbscrews located on the top of the instrument; these hold the
access door in place. The top probe mechanism and measurement chamber are revealed.
3.
Gently turn the probe adjustment clockwise, moving the probe block as far up as
possible, to ensure that it is high enough to accommodate any microplate with a height
up to 0.800" (or up to 1.250" for the FLx800TBEP model).
Note for FLx800TBEP model: To effectively read microplates using the top probe, a
plate carrier adapter is recommended (BioTek part number 7080522), to raise the
microplate and bring it closer to the probe. Microplates with heights up to 0.600" can
be read using this adapter.
Note for FLx800TBIDE model: For FLx800 models with a dispenser module, the
injector head moves up and down with the top probe.
4.
Open the plate carrier access door.
5.
Place the microplate on the plate carrier.
6.
Using your fingers, slide the plate carrier forward into the measurement chamber.
7.
Watch the carrier’s movement through the opening on top of the instrument. Once the
carrier is in the chamber, slide it to the right so that it is positioned beneath the probe
assembly.
8.
Gently turn the probe adjustment counterclockwise, lowering the probe block.
9.
When the probe block meets the plate, turn the adjustment clockwise one and one-half
turns.
FLx800 Operator’s Manual
22 | Chapter 2: Instrument Description
10.
Slide the plate carrier to the left, then draw it back out of the chamber.
11.
Replace the top probe access cover and four thumbscrews.
12.
Turn the instrument on to run a system test and home all axes. If the system test
completes successfully, plate reading can begin.
Lamp Assembly
The lamp assembly consists of a 20-watt Tungsten halogen lamp and bulb, with two cables
soldered on the back, and is accessible through the hinged door on the front of the instrument
(refer to Figure 1). The lamp is attached to a removable metal bracket that also holds the
condenser lens and heat absorber. The metal bracket is held in place with two screws running
down through the plate carrier compartment. The cables are plugged into a power source
located to the left of the Excitation filter wheel. The lamp assembly is shown in Figures 11
and 12 below. Replacement instructions are provided on the next page.
Lamp
Assembly
Figure 11: Removing the lamp assembly for replacement. Refer to Figure 12
below to ensure that the correct screws are removed.
Figure 12: This is a top-down view of the inside of the plate carrier compartment, showing the
orientation of the screws underneath the microplate carrier. Remove only the two leftmost screws to
remove the lamp assembly.
BioTek Instruments, Inc.
Lamp Assembly |
23
The bulb is expected to operate without replacement for a minimum of 1000 hours. The
intensity of the bulb will slowly drop over time until the instrument’s run-time self-check
detects a low signal and displays an error message. The lamp assembly should be replaced at
this time. A replacement lamp assembly is available from BioTek Instruments under the part
number 7080500.
Caution! Before replacing the lamp assembly, turn off and
unplug the instrument, then allow the lamp to cool down for a
minimum of 15 minutes before proceeding.
To replace the lamp assembly:
1.
Important! Turn off and unplug the instrument. Let the lamp cool down for at least 15
minutes before proceeding.
2.
Pull down the hinged door on the front of the instrument.
3.
Unplug the two cables connecting the lamp to its power source.
4.
Open the plate carrier compartment door.
5.
Gently slide the plate carrier into the reading chamber. Do not lift the carrier.
6.
Remove the two black screws located on the leftmost side of the plate carrier
compartment.
7.
Gently grasp the metal bracket housing the lamp and glass lenses, and then pull it
straight out of the lamp compartment. Do not touch the lenses!

Important! When removing the lamp from or
reattaching it to the metal bracket, do not touch the glass
lenses! Fingerprints on the condenser lens or heat
absorber may negatively affect performance.
8.
Remove the two thumbscrews that attach the lamp to the metal bracket.
9.
Remove the wire bracket from the lamp assembly.
10.
Place the wire bracket on the replacement lamp assembly.
11.
Seat the replacement lamp in its circular cutout, against the metal bracket.
12.
Replace the thumbscrews. When they are partially tightened, hold the metal bracket in
its upright position and gently twist the lamp so that the cables hang downward. Fully
tighten the thumbscrews.
13.
Slide the metal bracket into its slot in the lamp compartment.
14.
Replace the black screws in the plate carrier compartment.
FLx800 Operator’s Manual
24 | Chapter 2: Instrument Description
15.
Plug the lamp cables into the lamp’s power source. Either cable can be plugged into
either socket.
16.
Close the hinged door and the plate carrier door.
17.
Plug the instrument in and turn it on. If the system test passes, lamp replacement is
complete. If the test fails, note the error code and turn to Appendix B,
Troubleshooting.
BioTek Instruments, Inc.
Chapter 3
Installation
There is a certain sequence of events that must be followed to ensure
that the FLx800 and any peripheral devices are installed and
configured properly. This chapter describes the setup and configuration
tasks.
Unpacking and Inspection .................................................. 26
Unpacking and Inspecting the Dispenser Module ................... 30
Select the Operating Environment ....................................... 34
Connect Power ................................................................. 34
Install the Printer .............................................................. 35
Printers ....................................................................... 35
Turn on the Reader and Run a System Test.......................... 35
Check/Adjust the Filter Tables ............................................ 36
Install Software/Connect to Computer (Optional) .................. 38
Attach the Cable .......................................................... 38
Install Gen5 Software on the Host Computer ................... 38
Establish Communication .............................................. 38
Change the Baud Rate .................................................. 39
Configuring Global Default Options ................................. 40
Setting Up the Dispenser Module .................................... 40
Preparing the FLx800 for Shipment ..................................... 42
26 | Chapter 3: Installation
Unpacking and Inspection

Important! Save all packaging materials! If you need to
ship the reader to BioTek for repair or replacement, you must
use the original packaging materials. Using other forms of
commercially available packaging materials is not
recommended and can void the warranty.
If the original packaging materials have been damaged or lost,
contact BioTek. See Product Support & Service in
Appendix B for contact information.
See Preparing the FLx800 for Shipment at the end of this
chapter for complete shipping instructions.
 The instrument’s packaging is subject to change. If the instructions in this
section do not apply to the packaging materials you are using, please contact
BioTek’s Technical Assistance Center for guidance.
The FLx800 and its accessories are securely packaged inside custom-designed shipping
materials. This packaging should protect the instrument from damage during shipping.
Inspect the shipping box, packaging, instrument, and accessories for signs of damage.
If the shipping box has been damaged: Inspect the instrument for visible dents and
scratches as you unpack it.
If the reader is damaged: Notify the carrier and your BioTek sales representative. Keep
the shipping cartons and the packing materials for the carrier’s inspection. BioTek will arrange
for repair or replacement of your reader.
1.
Carefully open the top of the box, and remove any accessories.
2.
Remove the top end cap from the reader.
3.
Lift the reader out of the box, and place it on a level surface. Remove the reader
from the plastic bag.
4.
Remove the carrier shipping bracket and accompanying mounting hardware. Refer
to Figure 15.
5.
Attach the carrier shipping bracket to the back of the instrument using the
mounting hardware.
6.
Place all packing material back into the shipping box for reuse if the instrument
needs to be shipped again.
BioTek Instruments, Inc.
Unpacking and Inspection |
Declaration of Conformity
and factory data sheets
Operator’s Manual
Top end cap
Figure 13: Removing the top end cap and documentation
FLx800 Operator’s Manual
27
28 | Chapter 3: Installation
Power supply
Protective
plastic bag
Bottom end cap
Printer cable
Dust cover
Figure 14: Removing the instrument and accessories
BioTek Instruments, Inc.
Unpacking and Inspection |
Mounting hardware
Carrier shipping
bracket
Microplate carrier
Caution:
When removing the carrier
shipping block, do not
raise the carrier from the
instrument surface.
If the carrier is raised and
becomes fixed at an angle,
carefully slide the carrier
all the way to the right and
then lower it. Contact
BioTek TAC if any errors
are detected during the
System Test.
Carrier shipping bracket
and mounting hardware
Figure 15: Removing the microplate carrier shipping block
and storing it on the back of the instrument
FLx800 Operator’s Manual
29
30 | Chapter 3: Installation
Unpacking and Inspecting the Dispenser Module

Save all packaging materials. If you need to ship the dispense
module to BioTek for repair or replacement, you must use the
original materials. Using other forms of commercially available
packaging, or failing to follow the repackaging instructions,
may void your warranty.
During the unpacking process, inspect the packaging, module,
and accessories for shipping damage. If the dispense module is
damaged, notify the carrier and your BioTek representative.
Keep the shipping boxes and the packaging materials for the
carrier’s inspection. BioTek will arrange for repair or
replacement of your dispense module immediately.
Each dispense module is calibrated to perform with its specific
reader. Make sure the same serial number appears on both the
dispense module and the reader.
If applicable, perform these steps to unpack the dispense module. Refer to Figure 16 and
Figure 17 on pages 31 and 32.
1.
Open the shipping box. Remove the accessories box and foam insert that contains
the injector tubing and bottle holders.
2.
Lift out the module and place it on a level surface.
3.
Open the accessories box and remove its contents:
4.
•
Inlet tube
•
Outlet tube
•
Syringe
•
Cable
•
Syringe cover
•
Reagent bottle
•
Thumbscrew
•
Tip priming trough
•
Priming plate
Place all packaging materials into the shipping box for reuse if the dispense
module needs to be shipped.
BioTek Instruments, Inc.
Unpacking and Inspecting the Dispenser Module |
Foam insert
Accessories
box
Inner shipping
box
Foam insert
Outer shipping box
Figure 16: Removing the Dispenser Module and accessories from the outer shipping box
FLx800 Operator’s Manual
31
32 | Chapter 3: Installation
Shipping insert
Dispenser Module
Inner shipping
box
Figure 17: Removing the Dispenser Module box
BioTek Instruments, Inc.
Unpacking and Inspecting the Dispenser Module |
Dispenser
Module cable
Foam insert
Syringe cover
Inlet tube
Syringe
Outlet tube
Foam insert
Accessories
box
Figure 18: Removing the accessories
FLx800 Operator’s Manual
33
34 | Chapter 3: Installation
Select the Operating Environment
For best operation, install the FLx800 on a level, stable surface in an area where ambient
temperatures between 15°C (59°F) and 35°C (95°F) can be maintained.
The reader is sensitive to extreme environmental conditions. Be sure to avoid the following:
•
Excessive humidity: Condensation directly on the sensitive electronic circuits can
•
Excessive ambient light: Bright sunlight or strong incandescent light may affect the
•
cause the instrument to fail internal self-checks. The specified humidity range for this
instrument is from 10 to 85%, non-condensing.
instrument’s optics and readings, reducing its linear performance range.
Dust: Measurement values may be affected by extraneous particles (such as dust) in
the microplate wells. A clean work area is essential to ensure accurate readings.
Connect Power
1.
Connect the power cord to the external power supply.
2.
Locate the power inlet on the rear of the FLx800.
3.
Plug the rounded end of the power supply’s line cord into the power inlet.
4.
Plug the other end of the power cord into an appropriate power receptacle.
Warning! Power Rating. The FLx800 or power supply must
be connected to a power receptacle that provides voltage and
current within the specified rating for the system. Use of an
incompatible power receptacle may produce electrical shock
and fire hazards.
Warning! Electrical Grounding. Never use a plug adapter
to connect primary power to the FLx800 power supply. Use of
an adapter disconnects the utility ground, creating a severe
shock hazard. Always connect the power supply cord directly
to an appropriate receptacle with a functional ground.
BioTek Instruments, Inc.
Install the Printer |
35
Install the Printer
If the FLx800 will operate in standalone mode (that is, without BioTek’s Gen5 software running
on a host PC) connect a printer directly to the reader using the supplied cable.

Important! To avoid system instability, make sure the printer
and reader are turned OFF before connecting them.
1.
If the reader and/or printer are on, turn them off. Place the printer in a location adjacent
to the FLx800.
2.
Attach one end of the cable to the printer’s parallel port.
3.
Attach the other end of the cable to the parallel port on the FLx800.
4.
Make sure the securing screws on both ends of the cable are tightened.
5.
Turn on the reader, then turn on the printer.
Printers
The FLx800 supports printers using either HP’s PCL3 language, such as the HP DeskJet
series, or Epson’s LQ language. For the latest list of compatible printers call BioTek
Instruments’ Technical Assistance Center, or visit BioTek’s Web site and use the Search
feature. See Appendix B, Troubleshooting for contact information.
Turn on the Reader and Run a System Test
After installing the FLx800 and connecting the power supply, turn on the instrument to run a
system test. The on/off switch is located on the lower right side of the base.
The System Test begins with a check of the stepper motors and the analog power supplies, to
ensure that they have a proper input voltage level. The data flash checksum, motor axis, and
analog offset are then verified. Lastly, the PMT dark current, noise, and gain are checked to
ensure they fall within specific pass/fail criteria.
If an error is detected, the reader will “beep” and display an error code. If no errors are
detected, the reader will briefly display System Test Pass.
The power-up system test does not produce a printed results report. To run the test manually
and obtain a printout of the system test values, start at the Main Menu and press UTIL >
TESTS > SYSTEM. See System and Checksum Tests in Chapter 5 for more information.
FLx800 Operator’s Manual
36 | Chapter 3: Installation
•
If the test passes, the reader will beep once and the display will show the software’s
Main Menu, which will resemble the following:
READY
READ
•
10:15AM
11/16/10
DEFINE REPORT UTIL
If the test fails, the reader will beep repeatedly and the display will show an error
code. If this happens, write down the error code and then press the STOP key on the
keypad to stop the beeping. Look up the error code in Appendix B, Error Codes to
determine its cause.
•
If the problem is something you can fix, turn off the reader, fix the problem, and then
turn the reader back on. If the cause is not something you can fix, contact BioTek’s
Technical Assistance Center. See Appendix B for contact information.
Check/Adjust the Filter Tables
The FLx800 has two internal filter tables − Excitation and Emission − and their configurations
are maintained via the on-board software. Each filter table stores four sets of user-definable
wavelength and bandwidth values. When defining an assay, these wavelength/bandwidth sets
are presented as selectable options (see Define METHOD in Chapter 4 for more information).
Before using the FLx800, the filter tables must be configured to exactly match the filters installed
in the filter wheels. See Excitation and Emission Filter Wheels in Chapter 2 for information
on removing the filter wheels and determining which filters are installed.
To configure the Excitation and Emission filter tables:
1.
At the Main Menu, select UTIL.
READY
READ
2.
10:15AM
11/16/ 10
DEFINE REPORT UT IL
At the Select Utility Option screen, select SETUP.
SELECT UTILITY OPTION?
TESTS SETUP OUTPUT READ
3.
At the Edit Setup Information screen, select FILTER.
EDIT
DATE
4.
SETUP
TIME
INFORMATION
FILTER *MORE
The Filter Setup Selection screen will appear. Select EXCITATION to configure
the Excitation filter table, or EMISSION for the Emission filter table.
FILTER SETUP:
EXCITATION
EMISSION
BioTek Instruments, Inc.
Check/Adjust the Filter Tables |
5.
37
The Excitation/Emission (Lambda/Band) entry screen will appear.
EXCITATION(LAMBDA/BAND):
FILTER#1:
530/25
EMISSION
FILTER#1:
•
(LAMBDA/BAND):
OPEN
Use the numeric keys to enter the filter value in nanometers, and then the
bandwidth value.
 These values are printed on the side of each filter in the filter wheel.
See Excitation and Emission Filter Wheels in Chapter 2 for more
information.
•
Press the left or right arrow key to move the cursor left or right.
•
Press the STOP key to set the filter value to PLUG, indicating the presence of a
plug instead of a filter.
•
Press the CLEAR key to set the filter value to OPEN, indicating the absence of a
filter or plug.
•
Press the ENTER key to advance to the next filter entry screen.
 Pressing ENTER from the entry screen for Filter #4 returns to the Filter Setup
screen.

For luminescence assays:
A plug in the Excitation filter wheel is recommended, to
prevent any ambient light from entering the measurement
chamber. Note: During luminescence readings, the lamp is
automatically turned off.
An empty (open) location in the Emission filter wheel is
recommended, for optimal signal capture.
See Define METHOD in Chapter 4 for information on
selecting the Excitation and Emission filters to be used with
an assay.
6.
When all filter table modifications are complete, run the System Test.
•
To run the System Test, start at the Main Menu and select UTIL > TESTS >
SYSTEM.
 Any time a change is made to the Excitation and/or Emission filter table,
the System Test should be run.
FLx800 Operator’s Manual
38 | Chapter 3: Installation
Install Software/Connect to Computer (Optional)
Depending on the model, the FLx800 is equipped with one serial port or one serial port and
one USB port for communication with software (such as Gen5) running on an external
computer.
 The reader’s default communication parameters are: 9600 Baud Rate, 8
Data Bits, 2 Stop Bits, no parity. The baud rate can be changed, however,
Gen5 requires 9600. The other settings cannot be changed. See Chapter 4
for more information.
Attach the Cable
•
Connect one end to the appropriate port on the reader and the other end to the
appropriate port on the host computer.
Install Gen5 Software on the Host Computer

If applicable, install Gen5 on the host computer. There is a
certain sequence of events that must be followed to ensure
that the software is properly installed and configured. Please
follow the instructions provided in Gen5 Getting Started Guide
to install the software.
Establish Communication
 If your instrument came equipped with a USB port, refer to the
instructions that shipped with the USB Driver Software CD to install the
necessary drivers.
1.
Start Gen5 and log in if prompted. The default System Administrator password is
admin.
2.
Go to the Gen5 main screen:
•
Gen5 version 2.x users: From the Task Manager, select Setup > Go to System
Menu.
•
Gen5 version 1.x users: From the Welcome screen, select System Menu.
3.
Select System > Reader Configuration and click Add.
4.
Set the Reader Type to FLx800.
BioTek Instruments, Inc.
Install Software/Connect to Computer (Optional) |
5.
39
Set the Com Port to the computer’s COM port to which the reader is connected.
 If using the USB cable, the information can be found via the Windows Control
Panel, under Ports in the Hardware/Device Manager area of System Properties
(e.g., Serial Port (COM5)).
6.
Click Test Comm. Gen5 attempts to communicate with the reader. If the
communication attempt is successful, return to the Gen5 main screen.
If the communication attempt is not successful, try the following:
•
Is the reader connected to the power supply and turned on?
•
Is the communication cable firmly attached to both the reader and the computer?
•
Did you select the correct Reader Type in Gen5?
•
If applicable, did you install the USB driver software?
•
Make sure the reader display is at its Main Menu.
If you remain unable to get Gen5 and the reader to communicate with each other,
contact BioTek’s Technical Assistance Center.
Change the Baud Rate
 Gen5 requires the Baud Rate to be set at 9600.
If you need to change the Baud Rate:
1.
At the Main Menu, select UTIL.
READY
READ
2.
DEFINE
REPORT
UTIL
At the Select Utility Option screen, select SETUP.
SELECT UTILITY OPTION?
TESTS SETUP OUTPUT READ
3.
At the Edit Setup Information screen, select *MORE, then select RS232.
EDIT
DATE
SETUP
TIME
INFORMATION
FILTER *MORE
EDIT SETUP
RS232
INFORMATION
*MORE
FLx800 Operator’s Manual
40 | Chapter 3: Installation
4.
The Select Baud Rate screen will appear, showing the currently defined Baud Rate.
SELECT
9600
BAUD
19200
RATE:
38400
9600
VIEW
 Press the soft key under the desired rate.
 To view additional communication settings, select VIEW.
RS232 SETTINGS:NO PARITY
2 STOP BITS
8 DATA-BITS
 The Parity, Stop-Bits, and Data-Bits settings cannot be changed.
 Press the Main Menu key to return to the Main Menu.
Configuring Global Default Options
The FLx800 contains several global configurable options, such as date and time, report
output, and plate reading preferences. These options are accessed from the Main Menu by
selecting UTIL and include:
•
SETUP: Set the current date and time, as well as the date and time formats.
•
OUTPUT: Specify where plate data should be sent, to a printer, a computer, or to
both. Additional options include report format (Column and/or Matrix), and
whether or not to print standard curves.
•
READ: Enable or disable read-time prompting for Plate ID, Sample ID, and Sample
Count. Specify whether or not to read in Rapid mode
Setting Up the Dispenser Module
Perform the following steps to install and set up the Dispenser Module:
 Refer to Figures 3 through 6 in Chapter 2 for assistance.
1.
Turn off the FLx800.
2.
Place the Dispenser Module on top or to the left side of the reader.
3.
Screw the fitting of the inlet tube (8" or 20 cm with stainless steel probe, PN 7082121)
into the right side of the valve of the syringe drive on the Dispenser Module. Fingertighten the fitting only. Use no tools.
4.
Screw one fitting of the outlet tube (17" or 46 cm, PN 7082120) into the left side of the
valve of the syringe drive on the Dispenser Module. Finger-tighten the fitting only.
5.
Screw the other end of the outlet tube into the tubing port on the rear of the reader.
Finger-tighten the fitting only.
BioTek Instruments, Inc.
Install Software/Connect to Computer (Optional) |
6.
41
Install the syringe:
•
Carefully insert the white tip of the syringe plunger into the barrel of the syringe
all the way to the top of the syringe barrel.
•
Screw the threaded end of the syringe into the bottom threaded port of the valve of
the syringe drive. Finger-tighten only.
•
Carefully pull the knurled steel end of the syringe plunger until the end of the steel
is in the hole of the syringe drive bracket.
•
Pass the thumbscrew through the hole in the bottom of the syringe drive bracket
and thread it into the bottom of the steel end of the syringe plunger. Hold the
syringe plunger from rotating while tightening the thumbscrew. Finger-tighten
only.
7.
Plug the female end of the dispenser module cable into the dispenser port on the
left side of the Dispenser Module.
8.
Plug the other end of the dispenser module cable into the dispenser port at the
bottom of the rear panel of the reader.
9.
Install the tip priming trough in the right rear pocket of the microplate carrier.
10.
Turn on the FLx800. The instrument will perform its power-up self-test and initialize the
syringe drive of the Dispenser Module.
FLx800 Operator’s Manual
42 | Chapter 3: Installation
Preparing the FLx800 for Shipment

Important! Use the instrument’s original shipping container
and packaging material. Other forms of commercially
available packaging are not recommended and can void the
warranty. This shipping system was designed to be used no
more than five times. If the container is damaged and/or has
been used more than five times, contact BioTek for a new set of
shipping materials.
The instrument’s packaging design is subject to change over
time. If the instructions in this section do not appear to apply
to the packaging materials you are using, please contact
BioTek’s Technical Assistance Center for guidance.
Warning! If the reader has been exposed to potentially
hazardous material, decontaminate it to minimize the risk to
all who come in contact with the reader during shipping,
handling, and servicing. Decontamination prior to shipping is
required by U.S. Department of Transportation regulations
Before shipping the instrument and/or the Dispenser Module:
1.
Decontaminate the instrument and its accessories. See Appendix A,
Decontamination.
2.
Package the instrument and accessories as described below.
3.
Obtain a Return Materials Authorization (RMA) number from BioTek TAC (see
Appendix B, Troubleshooting for contact information). Mark this number on
the outside of the shipping box.
4.
Include instructions regarding what is required of the BioTek Service Department.
Clearly state whether the unit requires calibration, cleaning, periodic maintenance,
warranty work, and/or repair.
5.
Provide BioTek with the name and telephone number of a person who may be
contacted if questions arise.
BioTek Instruments, Inc.
Preparing the FLx800 for Shipment |
Repackaging the FLx800 and its accessories:
 Refer to Figures 13 through 15 while performing the following steps.
1.
Remove the 2 mounting screws that hold the shipping bracket in its storage
position on the back of the instrument.
2.
Install the carrier shipping block, using the 2 mounting screws.
3.
Remove the Tip Priming Trough from the right rear of the carrier and package it
with the other accessories.
4.
Place the instrument inside the plastic bag.
5.
Place the bottom foam end cap in the shipping box.
6.
Place the instrument in the box.
7.
Place the accessories along the sides of the instrument.
8.
Place the top foam end cap on the instrument.
9.
Close and seal the box.
Repackaging the Dispenser Module and its accessories:
 Refer to Figures 16 through 18 while performing the following steps.
1.
Remove the Inlet and Outlet Tubes and set them aside.
2.
Remove the syringe and set it aside.
3.
Disconnect the cable and set it aside.
4.
Remove the top end cap from the shipping box.
5.
Place the Dispenser Module in the bottom end cap in the shipping box.
6.
Place the Inlet Tube in its protective tube.
7.
Attach the fitting covers to the fittings of the Outlet Tube and place it in its
protective bag.
8.
Place the syringe in its protective box.
9.
Place the syringe and tubes in the pockets of the end cap.
10. Place the top end cap over the Dispense Module and seal the box.
FLx800 Operator’s Manual
43
44 | Chapter 3: Installation
BioTek Instruments, Inc.
Chapter 4
Operation
This chapter primarily discusses the operation of the FLx800 through
its front panel (2-line display and keypad). It also includes instructions
for getting started with Gen5.
Getting Started with Gen5 ................................................. 46
Introduction ..................................................................... 47
FLx800 Front Panel ........................................................... 47
The Keypad ................................................................. 48
System Startup ................................................................ 49
Main Menu ....................................................................... 50
DEFINE ............................................................................ 51
Selecting an Assay to Define .......................................... 52
Editing the Assay Name ................................................ 52
Define METHOD ........................................................... 53
Define MAP .................................................................. 62
Define FORMULA .......................................................... 74
Define CURVE .............................................................. 86
Panel Assays................................................................ 89
READ .............................................................................. 91
Selecting an Assay to Run ............................................. 92
Run-Time Prompts........................................................ 92
Beginning the Plate Read............................................... 94
REPORT ........................................................................... 95
UTILITY ........................................................................... 98
Setting the Date and Time ............................................. 98
Specifying Data Output and Reporting Options ................. 99
Selecting Read Options ............................................... 100
46 | Chapter 4: Operation
Getting Started with Gen5
These instructions briefly describe how to create and run an Experiment in Gen5. For more
information, or if the instructions below do not match what you see in Gen5, refer to the Gen5
Getting Started Guide and help system.
For Gen5 version 2.x:
1.
Start Gen5.
2.
If the Task Manager appears, select Read Now > New and skip to step 4.
Otherwise, select File > New Task from the main view.
3.
Select Read Now > New. Gen5 will open the procedure dialog. Skip to step 4.
For Gen5 version 1.x:
1.
Start Gen5. If the Welcome screen appears, select Read a Plate and skip to step 4.
Otherwise, select File > New Experiment from the main view.
2.
Select Default Protocol and click OK. Gen5 will open the Experiment workspace,
which includes the Protocol menu tree and Plate screen.
3.
Select Plate > Read or click the Read Plate icon. The Procedure dialog will open.
Go to step 4.
For any version:
4.
Select a Plate Type.
5.
Click Read to open the Read Step dialog.
6.
Select a Detection Method of Fluorescence or Luminescence.
7.
Select a Read Type of Endpoint or Area Scan (Fluorescence only).
8.
Select the number of Filter Sets to be used.
9.
Define other reading parameters as desired. Click the Help button for assistance.
10. When complete, click OK to return to the Procedure dialog.
11. Click OK to save and close the Procedure dialog.
•
Gen5 version 1.x only: The Plate Reading dialog will open. Enter any
desired information, place the plate on the carrier, and then click READ to
begin the plate read. If the Save As dialog opens, enter a File name, choose a
file location (Save in:) and click Save.
12. Click OK when the Load Plate dialog appears. The plate will be read.
•
To view the raw data results, use the Data drop-down arrow in the Plate
screen to select one wavelength. The results will be displayed for the
selected wavelength. Repeat, for other wavelengths.
•
To analyze, manipulate, or print results, Protocol parameters should be
defined. Refer to the Gen5 Help system for instructions.
BioTek Instruments, Inc.
Introduction |
47
Introduction
The FLx800’s front panel features a 2 x 24-character Liquid Crystal Display (LCD), and a 25key keypad. Through this interface you can create and modify fluorescence and luminescence
assays, read plates, store and print results, send results to an external computer, and more.
The FLx800 comes pre-loaded with “Custom” assays and/or with “Open” assays.
•
•
Custom assays are created by BioTek and allow you to easily select, modify, and run
several common assays directly from the front panel.
Open assays are defined with all possible parameters open for editing. You can
modify these parameters through the FLx800 front panel to create your own assays.
FLx800 Front Panel
READY
READ
Liquid Crystal
Display (LCD)
10:23AM
12/17/99
DEFINE REPORT UTIL
Menu options
SoftKeys (4)
Alphanumeric
keys
Special function
keys
Press the Shift key and an alphanumeric key
simultaneously to select a letter from A to H
Figure 19: FLx800 front panel, with 2x24-character LCD and keypad
FLx800 Operator’s Manual
48 | Chapter 4: Operation
The Keypad
The keypad has four SoftKeys, one below each selectable menu
option. Press a SoftKey to make a selection. For example, from the Main
Menu, press the leftmost SoftKey to select READ, the rightmost to select
UTIL.
Main
Menu
Options
ENTER
Previous
Screen
CLEAR
Exit the current screen and return to the Main Menu. Pressing Main
Menu while defining or modifying an assay automatically saves the
current settings.
Cycle through available options within a screen. For example, press
Options within the Select Assay Number screen to cycle through
the names of the on-board assays.
Pressing ENTER generally saves the current screen settings and
advances to the next screen in a series.
Pressing Previous Screen generally saves the current screen settings
and returns control to the screen most previously viewed.
Press CLEAR to reset a numeric value to 0, or to clear all characters
when editing an assay name. Tip: Press Shift + Clear at the Map
Generation screen to “clear” a previously defined manual map.

Move the cursor to the left in data-entry screens.

Move the cursor to the right in data-entry screens.
READ
Initiate a plate read.
STOP
Halt the read currently in progress.
BioTek Instruments, Inc.
System Startup |
49
System Startup
To turn on the FLx800, press the switch on the right side of the base. The FLx800 will perform a
System Test, displaying the screen shown below until initialization is complete. During this
time, all keys are inactive.
If the instrument fails the System Test, a beep will sound and an error code will display.
•
Refer to System and Checksum Tests in Chapter 5 for more information.
•
Refer to Appendix B, Troubleshooting to interpret error codes, and for information
on contacting BioTek Instrument’s Technical Assistance Center (TAC).
(Instrument ID)
System Self-Test...
FLx800 Operator’s Manual
50 | Chapter 4: Operation
Main Menu
Following successful power-up of the FLx800, the Main Menu appears:
READY
READ
08:12
DEFINE
12/17/10
REPORT UTIL
The Main Menu permits access to all reader functions:
•
READ: Choose a pre-defined assay for plate reading. Alternatively, press the key
labeled READ on the keypad (p. 91).
•
DEFINE: Create a new assay or modify an existing one. Definable parameters include
•
REPORT: Print a Result, Map, Assay, or List Report (p. 95).
•
UTIL: Run a System or Checksum Test. Set up various global configuration options
assay type, excitation filter, emission filter, sensitivity, well identifiers, plate mapping
options, formulas, and curve-fitting parameters (p. 51).
such as date and time, report output, and plate reading preferences (p. 98).
READY 03:27
11/07/99
READ DEFINE REPORT UTIL
SELECT UTILITY OPTION:
TESTS SETUP OUTPUT READ
PRINT REPORT:
RESULT MAP ASSAY
SELECT ASSAY NUMBER:
NAME: SSDNA QUANT
SELECT ASSAY NUMBER:
NAME: SSDNA QUANT
LIST
03
03
Figure 20: Options available from the Main Menu
BioTek Instruments, Inc.
DEFINE |
51
DEFINE
The Main Menu option DEFINE allows you to define the data acquisition and reduction
parameters for a new assay, or to modify a previously defined assay stored in memory.
1.
Start at the Main Menu and select DEFINE. The Select Assay Number screen will
appear.
SELECT ASSAY NUMBER:01
NAME:DNA QUANT
2.
Select an assay to define or modify, then press Enter. See Selecting an Assay to Define on
the next page for detailed instructions. The Edit Assay Name screen will appear.
NAME:
-
3.
DNA
/
QUANT
:
SPACE
(Optional) Edit the assay name, then press Enter. See Editing the Assay Name on the next
page for detailed instructions. The DEFINE menu will appear:
DEFINE:
METHOD MAP
FORMULA
CURVE
The following options are available within the DEFINE menu:
•
METHOD: Define the assay type (ex. Fluorescence or Luminescence), plate
geometry, Excitation and Emission filters, and sensitivity setting for data
acquisition (p. 53).
•
MAP: Specify the plate layout, using blanks, controls, standards, and/or samples.
Choose to map the plate manually, or let the software map it automatically (p. 62).
•
FORMULA: Define cutoff, transformation, validation, and/or general formulas.
•
CURVE: Specify a curve fit type and x/y axis types (lin/log). Specify whether or
Create variables to be used within formulas (p. 74).
not standard outliers can be edited, and then the method by which they will be
edited. Enable or disable the extrapolation feature (p. 86).
FLx800 Operator’s Manual
52 | Chapter 4: Operation
Selecting an Assay to Define
To select an assay to define or modify, start at the Main Menu and select DEFINE. The
Select Assay Number screen will appear:
SELECT ASSAY NUMBER:01
NAME:DNA QUANT
•
Use the numeric keys to enter the number of any predefined assay stored in
the reader’s memory, or the Option key to advance one assay at a time. The
cursor is positioned at the first editable field, and advances automatically. The
numeric range depends on the number of assays programmed in the reader’s
memory.
•
Press Enter to advance to the Edit Assay Name screen. You may change the
default assay name to a more descriptive one (see Editing the Assay Name):
 Clear: Clears the reader’s display.
 Main Menu: Returns the display to the Main Menu screen.
 Previous Screen: Returns the display to the previous screen.
 Enter: Saves the current settings and advances to the next screen.
Editing the Assay Name
Use the Edit Name screen to edit the name currently assigned to the assay. The assay name can
contain up to 16 alphanumeric characters.
NAME:
-
DNA
/
QUANT
:
SPACE
•
The cursor is positioned at the first editable field (ex. under “D”).
•
Use the Alpha and Numeric keys to change the Assay name.
•
Use the Option key to sequentially advance the character positioned above the
cursor. The characters will cycle through the alphabet (A-Z), with a space
following Z.
•
Use the Left and Right Arrow keys to move the cursor to the previous or next
editable field. The cursor will wrap around the edit field.
•
Use SoftKey 1, 2, 3, and 4 to select a dash, forward slash, colon, or space for
inclusion in the assay name.
BioTek Instruments, Inc.
DEFINE |
Define METHOD
The definition of METHOD parameters is essential for data acquisition.
1.
Start at the Main Menu and select DEFINE.
2.
Select the assay then press ENTER. The DEFINE options screen will appear.
DEFINE:
METHOD MAP
3.
FORMULA
CURVE
Select METHOD. The definable Method parameters include:
•
Assay type
•
Option to perform dual wavelength subtraction (automatically subtract the
results of filter set # 2 from the results of filter set # 1)
•
Plate type
•
Number of filter sets
•
Option to eject the plate between filter sets
•
For each filter set:
 Top or bottom optical probe (reader-dependent)
 Excitation filter
 Emission filter
 Samples per well
 Delay before sampling
 Delay between samples
 Sensitivity setting
 Automatic sensitivity selection options
•
First well location
•
Last well location
FLx800 Operator’s Manual
53
54 | Chapter 4: Operation
DEFINE
METHOD MAP FORMULA CURVE
B
ASSAY TYPE
DELAY BEFORE
SAMPLES
PLATE TYPE
DELAY BETWEEN
SAMPLES
SINGLE or DUAL
WAVELENGTH
SENSITIVITY
Value or AUTO
Value
AUTO
1
NUMBER OF
FILTERSETS
AUTO-SENSITIVITY
WELL LOCATION
2 or 3
EJECT BETWEEN
SETS?
END
A
EDIT FILTERSET #
1, 2, 3, or END
1, 2, or 3
AUTO-SENSITIVITY
MAXIMUM VALUE
Yes
MULTIPLE
FILTERSETS?
No
SELECT PROBE
A
EXCITATION
DEFINE
METHOD MAP FORMULA CURVE
EMISSION
SAMPLES PER WELL
B
Figure 21: Options available under Define METHOD
BioTek Instruments, Inc.
DEFINE |
55
Assay Type
Use the Assay Type screen to indicate whether this is a Fluorescence or Luminescence
assay. This information is primarily used to tailor the report formats, although if
LUMIN is specified in an assay, the reader will automatically turn off the lamp when
that assay is run.
ASSAY
FLUOR
•
•
TYPE: FLUOR
LUMIN
Select FLUOR to indicate a fluorescence assay.
Select LUMIN to indicate a luminescence assay.
Plate Type
The FLx800 is capable of taking measurements for several different plate formats. See
Technical Specifications, Microplates in Chapter 1 for detailed information on the
supported plate types.
PLATE
6
•
TYPE:
12
96
24
WELLS
*MORE
Press *MORE to cycle through the available options.
6:
6-well (2 x 3)
96: 96-well (8 x 12)
12: 12-well (3 x 4)
384: 384-well (16 x 24)
24: 24-well (4 x 6)
96H: 96-well Hellma Quartz (8 x 12)
48: 48-well (6 x 8)
96M: 96-well Metric (8 x 12, 9 mm well spacing)
Wavelength
The FLx800 provides the option to automatically subtract the results of Filter Set # 2
from the results of Filter Set # 1.
WAVELENGTH:
SINGLE DUAL
SINGLE
•
Select SINGLE to indicate no automatic subtraction of results. If SINGLE is
selected, 1, 2, or 3 filtersets may be specified.
•
Select DUAL to instruct the software to automatically subtract the results of
Filter Set # 2 from the results of Filter Set # 1. One set of results is printed.
 If DUAL is selected, exactly 2 filtersets must be specified.
FLx800 Operator’s Manual
56 | Chapter 4: Operation
Filtersets
A filterset contains important plate reading information, such as the Excitation and
Emission filters, optics position, sensitivity, and sampling options. The FLx800 can
perform endpoint fluorescence or luminescence reads using 1, 2, or 3 uniquely defined
filter sets.
ENTER NUMBER
FILTERSETS:
•
OF
2
Enter the number of filter sets (1, 2, or 3) to use with this assay.

There are many applications for the use of multiple filter sets,
including:
•
Assays that have more than one fluorophore per well.
•
Comparison of results among varying sensitivity settings
with otherwise identical filterset settings.
•
Comparison of results between the top and bottom optics
positions (if the reader is equipped with dual-optics). Select
two filter sets with identical settings, one for the top probe
and the other for the bottom probe.
•
Comparison of results among various sampling settings.
Eject Between Sets
If 2 or 3 filtersets are specified, the Eject Between Sets? option is presented.
EJECT
YES
BETWEEN
NO
SETS?
YES
•
Choose YES to eject the plate after each filterset read.
•
Choose NO to keep the plate in the chamber for all filterset reads.
BioTek Instruments, Inc.
DEFINE |

57
Set Eject Between Sets to YES to:
•
Perform an initial reading of a blank plate, followed by one
or two assay plates (depending on the number of filter sets
specified).
Note: Subtraction between plates must be done manually.
•
Provide the ability to add reagents to the same plate between
readings.
•
Enable the reading of up to 3 different plates with one assay.
Edit Filterset
If more than one filterset will be defined for this assay, the Edit Filterset screen will
appear.
EDIT
1
•
FILTERSET:
2
3
1
END
Select 1, 2, or 3 to define a particular filterset’s parameters.
 After defining each filterset, this screen will reappear.
•
Select END when all filtersets have been defined.
Optical Probe
Certain FLx800 models are equipped both a top and a bottom optical probe. If the
instrument is equipped with both probes, the Probe screen will appear.
PROBE:
TOP
BOTTOM
BOTTOM
•
Select TOP to use the top optical probe for measurement.
•
Select BOTTOM to use the bottom optical probe for measurement.
Excitation Filter
The Excitation filter is used to select the narrow band of light to which the sample will
be exposed.
FLx800 Operator’s Manual
58 | Chapter 4: Operation
EXCITATION:
380
485
PLUG
EXCITATION:
PLUG
485/20
*MORE
485/20
*MORE
•
The options presented for selection represent the current filter setup in the
Excitation filter table. See Configuring the Excitation and Emission Filter
Tables in Chapter 2 for more information.
•
Select a filter value, in nanometers. When a filter is selected, the current selection is
updated with the filter value and the filter’s corresponding bandwidth value, as
defined in the Excitation filter table.
 In the example on the preceding page, the current Excitation filter is 485/20 nm.
485 is the wavelength at peak transmittance. 20 represents the range of
wavelengths transmitted at one-half the peak transmittance.
•
A PLUG in the excitation wheel is typically used for luminescence to prevent
ambient light from entering the measurement chamber.
Emission Filter
The Emission filter is used to select the band of emitted light with the maximum
fluorescence signal, to be measured by the photomultiplier (PMT).
EMISSION:
460
528
EMISSION:
PLUG
PLUG
528/20
*MORE
528/20
*MORE
•
The options presented for selection represent the current filter setup in the
Emission filter table. See Configuring the Excitation and Emission Filter
Tables in Chapter 2 for more information.
•
Select a filter value, in nanometers. When a filter is selected, the current
selection is updated with the filter value and the filter’s corresponding
bandwidth value, as defined in the Emission filter table.
 In the example above, the current Emission filter is 528/20 nm. 528 is the
wavelength at peak transmittance. 20 represent the range of wavelengths
transmitted at one-half the peak transmittance, or 528 ± 10 nm.
•
OPEN indicates the absence of a filter in the filter wheel. This is typically used
for luminescence assays, to ensure optimal signal capture.
BioTek Instruments, Inc.
DEFINE |

59
The OPEN position should never be used for fluorescence. If the
FLx800 is used for luminescence, positions 3 and 4 should
always have a PLUG or filter.
Samples Per Well
The FLx800 can take from 1 to 255 measurements per well. The value reported for the
well represents the average of all measurements taken.
SAMPLES
PER WELL:
010
•
Enter the number of measurements to be taken for each well. The valid range is
from 1 to 255 measurements.
•
Usually, the more samples per well the better the CV’s, although selecting a large
number of samples per well typically results in only marginal improvement.
Consider a setting that represents the optimal combination of sensitivity and
speed. Note: The reader takes approximately 10 milliseconds to perform each
measurement.
Delay Before Samples
Preceding the measurement of a microwell, the plate carrier positions the microplate
so that the well sits directly above (or below) the optical probe. An optional delay can
be specified between the time the plate carrier stops moving and the time the
measurement is taken.
DELAY
BEFORE
•
SAMPLES:
0350
Enter the delay, in milliseconds. The valid range is from 10 to 2550 milliseconds,
in 10 ms increments.
Delay Between Samples
An optional delay between the measurements can be specified. For some assays, a
longer delay between samples may result in better CV’s.
DELAY
BETWEEN
•
SAMPLES:
001
Enter the delay, in milliseconds. The valid range is from 0 to 255 milliseconds,
in 1 ms increments.
FLx800 Operator’s Manual
60 | Chapter 4: Operation

Delays longer than 200 milliseconds may bleach the samples.
Sensitivity
“Sensitivity” is a term used when referring to the photomultiplier (the device that
measures the light emitted from the sample). The signal from the range of fluorescence
and luminescence assays can be very weak, very strong, or somewhere in between.
The sensitivity, or gain, of the photomultiplier should be adjusted for each filter set to
ensure that the signals from all wells fall within the appropriate dynamic range of 0 to
99,999 relative fluorescence or luminescence units.
SENSITIVITY:
AUTOMATIC
•
030
SELECT
Press SELECT then enter a value representing the voltage to apply to the PMT.
 Valid values include 0, and a value in the range 25 to 255.
•
Alternatively, select AUTOMATIC to let the FLx800 automatically select the most
appropriate sensitivity value for the assay (see Auto Sensitivity Selection on
the next page).
•
Tips for selecting the most appropriate sensitivity:
 Typical fluorescence assays using 96- (or fewer) well plates require sensitivities
between 50 and 100. Assays using 384-well or other higher-density plates will
likely require higher sensitivities due to narrower optical probes.
 Typical luminescence assays require sensitivities between 100 and 250.
 As the sensitivity setting increases, so will the measurement values. If many
wells are reported as out-of-range, indicating relative fluorescence/luminescence
values greater than 99,999, the sensitivity setting is probably too high.
 A sensitivity setting of 25 applies 200 volts to the PMT; this is usually too low for
typical assays (see Tips above). A setting of 255 applies 1000 volts. A setting of 0
applies no voltage to the PMT.
BioTek Instruments, Inc.
DEFINE |
99999
99999
RFU or LFU
RFU or LFU
RFU or LFU
99999
0
0
0
Range of samples
“Optimum” sensitivity
Samples fall within the
0 to 99999 RFU/RLU range.
Lowest values are above 0.
61
Range of samples
Range of samples
Sensitivity is too low
Sensitivity is too high
Some samples are
reported as 0 RFU/RLU.
Some samples reach or
exceed 99999 RFU/RLU.
Auto Sensitivity Selection
The FLx800 provides the Auto Sensitivity Selection feature as an alternative to the
more manual method of defining multiple filter sets with varying sensitivity settings.
See Sensitivity on the preceding page for information on enabling this feature.
If this feature is enabled, the FLx800 will perform several calibration measurements of
a user-specified target well on the microplate. With each reading, the sensitivity (gain)
of the photomultiplier is increased. The target well is monitored to ensure that it, and
hence the assay, does not exceed a user-specified maximum value. Measurements will
stop when the target well reaches the maximum value. The sensitivity that produced
the relative fluorescence/luminescence value closest to the maximum value (without
exceeding it) is chosen for the microplate read.

When the plate read is complete, the printed reports show the
chosen sensitivity.
Well Location
To assist the FLx800 with sensitivity selection, one well on the plate must be
designated for use with auto sensitivity calibration measurements.
AUTO-SENSITIVITY
AT WELL LOCATION:
•
A01
Using the alpha and numeric keys, specify the target well that represents the
maximum fluorescence or luminescence reading for the plate.
 It is very important that this well be the well furnishing the highest
signal. If not, other wells might overrange.
FLx800 Operator’s Manual
62 | Chapter 4: Operation
Auto-Sensitivity Maximum Value
Once the target well has been designated, a maximum relative fluorescence or
luminescence value must be assigned to the well.
AUTO-SENSITIVITY
MAXIMUM VALUE:
•
70000
The valid range is from 00010 to 90000 relative fluorescence/luminescence
units.
 Auto-Sensitivity Maximum Values above 70,000 are not recommended
unless the selected well is the highest well for the entire microplate.
Define MAP
To configure the layout of the plate using the MAP parameters:
1.
Start at the Main Menu and select DEFINE.
2.
Select the assay then press ENTER. The DEFINE options screen will appear.
DEFINE:
METHOD MAP
3.
FORMULA
CURVE
Select MAP. The definable Map parameters include:
•
Automatic or Manual Map Generation
•
Mapping Direction
•
Replicate Direction
•
Blank Map Selection
•
Blanking Constant
•
Number of Blanks
•
Location of Blanks
•
Number of Standards
•
Number of Standard Replicates
•
Averaging of Standards
•
Concentration and Location of Standards
•
Number of Controls
•
Control Type Definition
BioTek Instruments, Inc.
DEFINE |
•
Number of Control Replicates
•
Control Location
•
Number of Samples
•
Number of Sample Replicates
•
Sample Location
63
DEFINE
METHOD MAP FORMULA CURVE
A
B
MAP GENERATION
AUTO or MANUAL
LOCATION OF BLANKS
(MANUAL map only)
NUMBER OF
CONTROLS
MAPPING
DIRECTION
NUMBER OF
STANDARDS
SELECT CONTROL
IDENTIFIERS
REP DIRECTION
(AUTO map only)
NUMBER OF STANDARD
REPLICATES
NUMBER OF CONTROL
REPLICATES
START MAPPING AT
WELL LOCATION
AVERAGE STANDARDS?
LOCATION OF CTRLS
(MANUAL map only)
BLANK MAP
ENTER STANDARD
CONCENTRATIONS
NUMBER OF SAMPLES
NUMBER OF BLANKS
ENTER STD LOCATIONS
(MANUAL map only)
NUMBER OF SAMPLE
REPLICATES
BLANKING CONSTANT
REUSE STANDARD
CURVE?
LOCATION OF SAMPLES
(MANUAL map only)
A
B
DEFINE
METHOD MAP FORMULA CURVE
Figure 22: Options available under Define MAP
FLx800 Operator’s Manual
64 | Chapter 4: Operation
Map Generation
“Map Generation” represents the method by which blanks, controls, standards,
and/or samples are assigned to specific locations on the plate.
MAP GENERATION:
AUTO
MANUAL
MANUAL
•
Select AUTO to instruct the software to automatically generate a Plate Map after
the blanks, controls, standards, and/or samples have been defined.
•
Select MANUAL to indicate that the well assignments will be performed manually
(by the user) at Define and/or Run time.

Note: Press Shift + Clear at the MAP GENERATION
screen to “clear” a previously defined manual map.
Mapping Direction
The Mapping Direction is the direction (Down or Across) in which blank, control,
standard, or sample groups will be mapped on the plate.
MAPPING DIRECTION:DOWN
DOWN ACROSS
•
Select DOWN to map groups down the plate columns.
•
Select ACROSS to map groups across the plate rows.
•
For example, if the plate contains one blank (BLK), one negative control (NC),
one positive control (PC), and samples with one replicate:
 DOWN maps the wells as A1:BLK, B1:NC, C1:PC, D1:SMP1,
E1:SMP2, and so on.
 ACROSS maps the wells as A1:BLK, A2:NC: A3:PC, A4:SMP1,
A5:SMP2, and so on.
•
Additional examples of mapping directions are shown in Figure 23.
BioTek Instruments, Inc.
DEFINE |
Replicate Direction
The “Replicate Direction” is the direction (Down or Across) in which blank, control,
standard, or sample replicates will be mapped on the plate.
REP DIRECTION:
DOWN ACROSS
DOWN
•
Select DOWN to map replicates down the plate columns.
•
Select ACROSS to map replicates across the plate rows.
•
For example, if the plate contains two blanks (BLK), two negative controls (NC),
two positive controls (PC), and samples in duplicate:
 DOWN maps the wells as A1/B1:BLK, C1/D1:NC, E1/F1:PC,
G1/H1:SMP1, and so on.
 ACROSS maps the wells as A1/A2:BLK, A3/A4:NC, A5/A6:PC,
A7/A8:SMP1, and so on.
•
Additional examples of mapping directions are shown on the next page.
FLx800 Operator’s Manual
65
66 | Chapter 4: Operation
Map Direction DOWN, Rep Direction DOWN:
A
B
C
D
E
F
G
H
1
STD1
STD1
STD2
STD2
STD3
STD3
STD4
STD4
2
STD5
STD5
PC
PC
NC
NC
SMP1
SMP1
3
SMP2
SMP2
SMP3
SMP3
SMP4
SMP4
SMP5
SMP5
4
SMP6
SMP6
SMP7
SMP7
SMP8
SMP8
SMP9
SMP9
5
SMP10
SMP10
SMP11
SMP11
SMP12
SMP12
SMP13
SMP13
6
SMP14
SMP14
SMP15
SMP15
SMP16
SMP16
SMP17
SMP17
7
SMP18
SMP18
SMP19
SMP19
SMP20
SMP20
SMP21
SMP21
8
SMP22
SMP22
SMP23
SMP23
SMP24
SMP24
SMP25
SMP25
9
SMP26
SMP26
SMP27
SMP27
SMP28
SMP28
SMP29
SMP29
10
SMP30
SMP30
SMP31
SMP31
SMP32
SMP32
SMP33
SMP33
11
SMP34
SMP34
SMP35
SMP35
SMP36
SMP36
SMP37
SMP37
12
SMP38
SMP38
SMP39
SMP39
SMP40
SMP40
SMP41
SMP41
6
STD3
SMP2
SMP8
SMP14
SMP20
SMP26
SMP32
SMP38
7
STD4
SMP3
SMP9
SMP15
SMP21
SMP27
SMP33
SMP39
8
STD4
SMP3
SMP9
SMP15
SMP21
SMP27
SMP33
SMP39
9
STD5
SMP4
SMP10
SMP16
SMP22
SMP28
SMP34
SMP40
10
STD5
SMP4
SMP10
SMP16
SMP22
SMP28
SMP34
SMP40
11
PC
SMP5
SMP11
SMP17
SMP23
SMP29
SMP35
SMP41
12
PC
SMP5
SMP11
SMP17
SMP23
SMP29
SMP35
SMP41
6
SMP10
SMP11
SMP12
SMP13
SMP14
SMP15
SMP16
SMP17
7
SMP18
SMP19
SMP20
SMP21
SMP22
SMP23
SMP24
SMP25
8
SMP18
SMP19
SMP20
SMP21
SMP22
SMP23
SMP24
SMP25
9
SMP26
SMP27
SMP28
SMP29
SMP30
SMP31
SMP32
SMP33
10
SMP26
SMP27
SMP28
SMP29
SMP30
SMP31
SMP32
SMP33
11
SMP34
SMP35
SMP36
SMP37
SMP38
SMP39
SMP40
SMP41
12
SMP34
SMP35
SMP36
SMP37
SMP38
SMP39
SMP40
SMP41
6
PC
PC
SMP11
SMP11
SMP23
SMP23
SMP35
SMP35
7
NC
NC
SMP12
SMP12
SMP24
SMP24
SMP36
SMP36
8
SMP1
SMP1
SMP13
SMP13
SMP25
SMP25
SMP37
SMP37
9
SMP2
SMP2
SMP14
SMP14
SMP26
SMP26
SMP38
SMP38
10
SMP3
SMP3
SMP15
SMP15
SMP27
SMP27
SMP39
SMP39
11
SMP4
SMP4
SMP16
SMP16
SMP28
SMP28
SMP40
SMP40
12
SMP5
SMP5
SMP17
SMP17
SMP29
SMP29
SMP41
SMP41
Map Direction ACROSS, Rep Direction ACROSS:
A
B
C
D
E
F
G
H
1
STD1
NC
SMP6
SMP12
SMP18
SMP24
SMP30
SMP36
2
STD1
NC
SMP6
SMP12
SMP18
SMP24
SMP30
SMP36
3
STD2
SMP1
SMP7
SMP13
SMP19
SMP25
SMP31
SMP37
4
STD2
SMP1
SMP7
SMP13
SMP19
SMP25
SMP31
SMP37
5
STD3
SMP2
SMP8
SMP14
SMP20
SMP26
SMP32
SMP38
Map Direction DOWN, Rep Direction ACROSS:
A
B
C
D
E
F
G
H
1
STD1
STD2
STD3
STD4
STD5
PC
NC
SMP1
2
STD1
STD2
STD3
STD4
STD5
PC
NC
SMP1
3
SMP2
SMP3
SMP4
SMP5
SMP6
SMP7
SMP8
SMP9
4
SMP2
SMP3
SMP4
SMP5
SMP6
SMP7
SMP8
SMP9
5
SMP10
SMP11
SMP12
SMP13
SMP14
SMP15
SMP16
SMP17
Map Direction ACROSS, Rep Direction DOWN:
A
B
C
D
E
F
G
H
1
STD1
STD1
SMP6
SMP6
SMP18
SMP18
SMP30
SMP30
2
STD2
STD2
SMP7
SMP7
SMP19
SMP19
SMP31
SMP31
3
STD3
STD3
SMP8
SMP8
SMP20
SMP20
SMP32
SMP32
4
STD4
STD4
SMP9
SMP9
SMP21
SMP21
SMP33
SMP33
5
STD5
STD5
SMP10
SMP10
SMP22
SMP22
SMP34
SMP34
Figure 23: Examples of Mapping Directions
BioTek Instruments, Inc.
DEFINE |
67
Start Mapping at Well Location
Enter the location of the well that will be the starting point for automatic mapping.
START MAPPING
AT WELL LOCATION:
•
A01
Use the NUMERIC and ALPHA keys to enter a letter or number at the cursor
location. The valid entry range is from A01 to the last well on the plate, depending
on the plate type and the number of blanks, standards, controls, and/or samples
defined in the assay.
Blank Map
There are several blanking methods to choose from.
BLANK
AIR
MAP: FULL
FULL
CONST
*MORE
•
AIR performs an initial reading “on air” just prior to the plate read, and uses that
•
FULL enables a single blank well or an average of blank wells to be subtracted
•
CONST (Constant) allows entry of a user-specified fluorescence/luminescence
•
ROW enables a single blank well or an average of blank wells to be selected for
•
COLUMN enables a single blank well or an average of blank wells to be selected for
reading as the blank value. This value is subtracted from each well on the plate.
from each well on the plate.
value. This value will be subtracted from each well on the plate.
each row. Use manual mapping to position blanks, standards, controls, and
samples.
each column. The blank (or average) value will be subtracted from other wells in
the column. Use manual mapping to position blanks, standards, controls, and
samples.
•
P-ACROSS enables a blank in every even-numbered column to be subtracted from
the well to the left of it in every odd column. Use manual mapping to set up the
appropriate map by placing the standards, controls, and samples in only the odd
columns.
•
P-DOWN enables a blank in the B, D, F, H, etc. rows to be subtracted from the well
above in the A, C, E, G, etc. rows. Use manual mapping to set up the appropriate
map by placing the standards, controls, and samples in rows A, C, E, G, etc.
FLx800 Operator’s Manual
68 | Chapter 4: Operation
Blanking Constant
If the CONST blanking method is selected, a Blanking Constant must be defined. This
value will be subtracted from each well on the plate.
ENTER
BLANKING
•
CONSTANT:
10000
Use the numeric keys to enter the value. The valid entry range is 0 to 99999
relative fluorescence/luminescence units.
Number of Blanks
If the Full, Column, or Row blanking method is selected, enter the number of blank
wells on the plate.
ENTER NUMBER
BLANKS:
•
OF
02
Use the numeric keys to enter the number of blank wells. The valid entry range
is 0 to 48 wells.
Blank Location
The Blank Location screen is presented if blank wells are defined and manual map
generation is selected.
ENTER THE
BLANK# 1:
LOCATION
OF
A01
•
Use the numeric and alpha keys to enter the location of the first Blank well.
•
Press Enter to define subsequent Blank well location(s).
BioTek Instruments, Inc.
DEFINE |
69
Number of Standards
For assays requiring standards, begin by entering the number of standard groups.
ENTER NUMBER
STANDARDS:
•
OF
04
Use the numeric keys to enter the number of standard groups.
 Enter 00 to indicate no standards.
•
The valid entry range depends on the selected curve fit method.
The maximum number of standards for all curve fit methods is 12.
The minimum numbers of standards are:
 4 for 2-P, 4-P, cubic, cubic spline
 3 for quadratic
 2 for linear, point-to-point
 Note: If the NUMBER OF STANDARDS setting is modified, the number of
standard replicates (see below) automatically defaults to 01.
Number of Standard Replicates
The Standard Replicates entry screen is presented if the number of standard groups is
greater than 0.
ENTER NUMBER OF
STANDARD REPLICATES:
•
02
Use the numeric keys to enter the number of standard replicates. The valid entry
range is from 1 to 8 replicates.
 The software will automatically verify that the number of replicates, multiplied
by the number of standards, does not exceed the number of wells on the plate.
Average Standards
If the Number of Standard Replicates setting is greater than 01, the Average Standards
screen is presented.
AVERAGE
YES
STANDARDS?
NO
YES
•
Select YES to average the replicates for each standard group, and then use the
group averages when calculating the standard curve.
•
Select NO to use the individual standard replicates when calculating the
standard curve.
FLx800 Operator’s Manual
70 | Chapter 4: Operation
Standard Concentrations
Use the Standard Concentration screen to enter the predicted concentration value for
each standard group. If manual map generation is selected, the replicate locations
must also be defined.
CONCN OF
STD1:00001
LOCATION
REP# 1:B01
•
Use the NUMERIC, ALPHA, and DECIMAL POINT keys to enter the standard
concentration values. The valid entry range is .00001 to 999999. The entry cannot
exceed 6 characters, including the decimal point.
•
If automatic mapping is selected, each replicate’s location is available for viewing
only. Pressing ENTER advances to the concentration value entry for the next
standard.
•
If manual mapping is selected, the location must be defined. Pressing ENTER from
the standard concentration entry moves the cursor to the Location field. Pressing
ENTER from the Location field advances to the concentration value entry for the
next standard.
Reuse Standard Curve
The FLx800 has the ability to reuse a standard curve that has already been established.
This option is available for assay numbers 30 through 55 only.
REUSE
YES
•
STANDARD
NO
CURVE?YES
Select YES so that, after a plate is read with this assay, a SAVE STANDARD
CURVE? YES/NO prompt will be presented.
 See Saving a Standard Curve below for more information.
•
Select NO to prohibit the appearance of the SAVE STANDARD CURVE? run-time
prompt.
Saving a Standard Curve
After the assay has been run, the results have been calculated, and the reports have
been generated, the reader will present the Save Standard Curve? prompt.
SAVE
YES
STANDARD
NO
CURVE?
YES
•
Select YES to store the curve for use at a later time.
•
Select NO to discard the curve.
BioTek Instruments, Inc.
DEFINE |
71
Reusing a Stored Curve
The next time this assay is run, the reader will present the Standards on Plate?
prompt.
STANDARDS ON
YES
NO
PLATE?
NO
•
Select YES to generate a new standard curve.
•
Select NO to calculate concentrations using the stored standard curve.
 If Auto mapping was originally used to map the standards, blanks, controls
and samples defined for this assay, the map will be automatically regenerated
without the standards, beginning in well xxx (where xxx was chosen as the
Starting well in the map, typically well A01).
 If Manual mapping was used to map the plate, the map is NOT regenerated
and the reader will NOT produce results for the well positions that originally
were standards. Auto mapping is recommended, if the standards curves
will be routinely re-used.
Limitations on the Reuse of Standard Curves
• The reuse of standard curves can only be done in assay positions 30 through 55.
Each of these positions can only store one standard curve.
•
In order for a standard curve to be reused, the assay name from the stored curve
and the name of the assay being run must be identical.
•
Standard curves cannot be reused on Panel assays.
•
Standard curves will be stored with the Assay Name, Standard Concentrations,
Replicate Counts, and Optical Densities for each standard replicate.

Important: If custom assays were pre-programmed at BioTek,
they may not be configured to reuse standard curves. Contact
BioTek for more information.
Number of Controls
For assays requiring controls, begin by entering the number of control groups.
ENTER NUMBER
CONTROLS:
FLx800 Operator’s Manual
OF
02
72 | Chapter 4: Operation
•
Use the NUMERIC keys to enter the Number of Control groups in the assay. For
example, if the assay requires one or more positive control wells and one or more
negative control wells, enter 02.
•
The valid entry range depends on the number of locations on the plate that are
undefined. The maximum number of control groups is 8.
Control Type
After defining the number of controls for this assay, select the types of controls to use.
CONTROL# 1:
PC
NC
HPC
PC
*MORE
•
Choose one control identifier for each type of control in your assay. The available
options are: Positive Control, Negative Control, High Positive Control, Low
Positive Control, CTL1, CTL2, CTL3, CTL4.
•
After choosing an identifier for Control #1, press Enter to choose the identifier for
the next control.
Number of Control Replicates
The Number of [Control] Replicates entry screen is presented if the number of control
groups is greater than 0.
ENTER NUMBER OF
REPLICATES OF PC:
02
•
The well ID associated with Control #1 appears first. Press Enter to advance to the
next control.
•
Use the numeric keys to enter a value for Number of [Control] Replicates.
•
The valid entry range is from 1 to 12 replicates. The software automatically
performs a check to ensure the number of replicates, multiplied by the number of
controls, does not exceed the number of undefined wells remaining on the plate.
Location of Controls
If mapping is manual and controls are defined, the locations for each control replicate
must be specified.
CONTROL#
TYPE:PC
•
1:
LOCATION
REP# 1:B01
Use the numeric and alpha keys to enter the well location for Rep #1 of Control
#1. Press Enter to advance to the next replicate or control group.
BioTek Instruments, Inc.
DEFINE |
73
Number of Samples
The number of sample groups on the plate can be defined here, and/or it can be defined at
run-time if UTIL > READ > PROMPT FOR SAMPLE COUNT? is set to YES. See
Selecting Read Options in Chapter 4 for more information.
ENTER NUMBER
SAMPLES:
OF
24
•
Use the numeric keys to enter the number of sample groups on the plate.
•
The range is 0 to the number of undefined well locations remaining on the plate.
For example, if there are no controls, blanks, or standards defined on a 96-well
plate, the maximum number of samples is 96, and the minimum number of
samples is 1.
Number of Sample Replicates
After the number of sample groups is specified, the Number of Sample Replicates entry
screen is presented.
ENTER NUMBER OF
SAMPLE REPLICATES:
02
•
Use the numeric keys to enter the number of sample replicates.
•
The valid entry range is from 1 to 12 replicates. The software automatically
performs a check to ensure that the number of replicates multiplied by the number
of samples does not exceed the number of undefined wells remaining on the plate.
Sample Location
If mapping is manual and samples are defined, the locations for each sample replicate
must be specified.
SAMPLE#
•
1
LOCATION
REP# 1:D02
Use the numeric and alpha keys to enter the well location for Rep #1 of Sample
Group #1. Press Enter to advance to the next replicate or sample group.
FLx800 Operator’s Manual
74 | Chapter 4: Operation
Define FORMULA
The FLx800 supports 4 types of formulas (Cutoff, Transformation, Validation, and
General), as well as the ability to program variables for use within formulas. Up to 3 types
of Validation formulas may be defined (Blank, Control, and Assay Validation).
To define formulas:
1.
Start at the Main Menu and select DEFINE.
2.
Select the assay then press ENTER. The DEFINE options screen will appear.
DEFINE:
METHOD MAP
FORMULA
CURVE
3.
Select FORMULA. The Select Formula Type screen will appear.
4.
Figure 24 is a flowchart showing all possible screens found under Define
FORMULA.
Calculation Structure
During data reduction, formulas are processed in the order shown below. The number of
permitted formulas of each type is shown as well.
•
Blank Validation
0-1
•
Control Validation
0-4
•
Assay Validation
0-4
•
Transformations
0-1
•
Cut-off Formulas
0-1
•
Curve Fit Analysis
(if a curve fit method is defined)
•
General Formulas
0-4
BioTek Instruments, Inc.
DEFINE |
DEFINE
METHOD MAP FORMULA CURVE
SELECT FORMULA TYPE *
CUTOFF TRANS VAL TRANS-VAR
ENTER FORMULA
(0 to 1)
ENTER FORMULA
(0 to 1)
SCOPE VARIABLE
SMP or R U
RU
SMP
ENTER GREYZONE %
ENTER FORMULA
SELECT TYPE
CTRL ASSAY BLK
SPECIFY CALL
ENTER FORMULA
(0 to 4)
# OF CTRLS REQUIRED
TO BE VALID
ENTER FORMULA
(0 to 4)
ENTER FORMULA
(0 to 1)
# OF BLKS REQUIRED
TO BE VALID
Figure 24: Options available under Define FORMULA
FLx800 Operator’s Manual
75
76 | Chapter 4: Operation
Formula Type
The FLx800 supports 4 types of formulas, as well as the ability to program variables for use
within Transformation formulas.
SELECT
CUTOFF
FORMULA TYPE:
TRANS
VAL
*MORE
SELECT FORMULA TYPE:
GENERAL TRANS-VAR
*MORE
•
•
CUTOFF formulas are used to classify results. During data reduction, results are
evaluated against the cutoff formulas and each well is assigned a user-specified
label (POS, NEG, or EQUIV).
TRANSformation formulas are applied to the fluorescence/luminescence data in
preparation for further data reduction and/or curve fit calculation.
•
VALidation formulas can be used to determine whether or not blanks and/or
controls are valid. In addition, Assay Validation formulas can be used to determine
whether or not the entire assay should be considered valid.
•
GENERAL formulas are calculated after all other calculations are complete, and the
results are not used in further data reduction.
 General formulas are not currently supported by the FLx800 on-board
software.
•
The TRANS-VAR option allows you to define a variable to be used in
transformation formulas.
BioTek Instruments, Inc.
DEFINE |
77
Formula Entry
After the formula type is selected, the Formula Entry screen appears. Each formula can
contain a maximum of 24 characters. Spaces are not necessary.
 In formulas, RU is a “generic” term used to represent either the Relative
Fluorescence Unit or the Relative Luminescence Unit, depending on the
assay type.
•
FORMULA#1:
MATH OTHER
MAP
FUNCTN
PC;X>50000
MATH OTHER
MAP
FUNCTN
After a moment, the FORMULA#1: prompt disappears and the formula can be
entered. Use the options found under MATH, OTHER, MAP, and FUNCTN to
“build” the formula.
 To cycle through the available Math, Other, Map, or Function options, continue
to press the appropriate SoftKey. For example, press the MATH SoftKey several
times to see +, -, *, /, %, =, etc. When the desired option appears, press the
RIGHT ARROW key to select it and advance to the next editable field.
 Press the LEFT ARROW key to move the cursor to the left.
 Press CLEAR to delete the item above the cursor.
 When a formula is complete, press ENTER to continue.
•
Select MATH to insert a mathematical symbol such as +, %, or <=.
•
Select OTHER to insert an opening “ (” or closing “) ” parenthesis, or logical
operators AND or OR.
•
Select MAP to insert a well ID such as BLK;x or NC;1.
•
Select FUNCTN to insert a mathematical function such as LOG or SQRT.

In the example shown above, the validation criteria states that
the mean (indicated by ;x) of the PC wells must be greater than
50000 to be considered valid. Your validation criteria will be
different. See additional examples on the next page.
The reader software checks the formulas for errors during data
reduction. A syntax error in a formula will result in a “Syntax”
or “Token Error” on results reports.
FLx800 Operator’s Manual
78 | Chapter 4: Operation
MATH
The following mathematical symbols can be used in formulas:
+
Addition
==
-
Subtraction
*
Multiplication
/
Division
<
%
Percent
<=
>
>=
Equal to
Greater than
Greater than or equal to
Less than
Less than or equal to
OTHER
The following additional symbols can be used in formulas:
(
Left parenthesis
)
Right parenthesis
AND
OR
Logical AND
Logical OR
MAP
The available MAP options depend on the formula type and the current plate map. MAP
options resemble BLK;x (mean of the blank wells), NC;1 (the first NC well), or RU (every
well).
FUNCTION
The following functions can be used in formulas:
LOG10
Log Base 10
ALOG
Anti Log
ALOG10
Anti Log Base 10
LOG
Log
Absolute Value
SQRT
Square Root
AB
PWR
Power
EXAMPLES
LOG10- Log Base 10Log10 2 = 0.301029995
ALOG- Anti LogALOG (0.69314718) = 2
ALOG10- Anti Log Base 10ALOG10 (0.30102995) = 2
LOG- LogLOG 2 = 0.69314718
AB- Absolute ValueAB (-1) = 1
SQRT- Square RootSQRT 2 = 1.4142
PWR- Power(10 PWR 2) = 100
BioTek Instruments, Inc.
DEFINE |
79
Validation Formulas
Validation formulas can be used to determine whether or not blanks and/or controls are
valid. In addition, Assay Validation formulas can be used to determine whether or not the
entire assay should be considered valid.
See Formula Type on page 76 for instructions on selecting an assay and accessing the
Select Validation Type screen.
SELECT VALIDATION
CONTROL
ASSAY
TYPE:
BLANK
Control and Blank Validation Formulas
Blank Validation is used to ensure that the RU values for the blank replicates, or for the
blank mean, meet a certain criteria. Control Validation serves the same purpose as Blank
Validation, but apply to the control replicates or control mean. If the criteria are not met,
results are considered suspect, and the message RESULTS INVALID! Blank (or
Control) validation failed appears on results reports.
•
One blank validation formula can be defined.
•
Up to 4 control validation formulas can be defined.
•
Define the plate map (via DEFINE > MAP) before creating blank or control
validation formulas.
•
Blank/Control validation can be performed on individual replicates (BLK, PC), or
on the group mean (BLK;x, NC;x).
Examples:
If an assay protocol states that:
•
Each Blank well should have a RU value less than 15000. The formula is:
BLK<15000
•
Each Positive Control replicate must fall within the RU range of 50000 to 75000.
This can be accomplished with one formula:
•
PC>50000 and PC<75000, or with two separate formulas:
•
PC>50000 and PC<75000
•
The Negative Control mean must have a RU value less than 20000. The formula
is: NC;x<20000
FLx800 Operator’s Manual
80 | Chapter 4: Operation
Number of Required Blanks / Controls
When a blank or control validation formula is defined, enter the number of blanks or
controls that must meet the criteria established by that formula.
PC:
NUMBER OF VALID
REPLICATES REQUIRED:
01
•
Use the NUMERIC keys to enter the number of blanks or controls that are required
to be found valid in order for the results to be valid. For example, if an assay states
that 2 out of 3 PC wells must be valid, enter 02.
•
The range is 1 through the number of defined replicates of the blank or control.
Assay Validation Formulas
Assay Validation formula(s) establish a set of criteria used to determine whether or not an
assay can be considered valid. If the criteria are not met, results are considered suspect,
and the message “RESULTS INVALID! Assay validation failed” appears on results reports.
•
Up to 4 assay validation formulas can be defined.
•
Define the plate map (via DEFINE > MAP) before creating assay validation
formulas.
Examples:
If an assay protocol states that in order for the assay to be valid:
•
The mean of the negative controls must be less than 25000. The formula is:
NC;x<25000
•
The mean of the positive controls must be greater than the mean of the negative
controls. The formula is:
PC;x>NC;x
BioTek Instruments, Inc.
DEFINE |
81
Transformation Formulas
Transformation formulas can be used to transform raw or blanked
fluorescence/luminescence data in preparation for further data reduction, including curve
fit analysis.
See Formula Type on page 76 for instructions on selecting an assay and accessing the
Transformation Formula definition screen.
•
If a blanking method is selected in the assay, transformation formulas are applied
to the blanked fluorescence/luminescence values; otherwise, they are applied to
the raw data. Turn to page 84 to review the results calculation structure.
•
One transformation formula may be defined per assay.
•
A transformation formula can be simple (ex. RU*100 to multiply all wells on the
plate by 100), or more complex with the inclusion of a pre-defined Transformation
Variable (see TVAR, below).
Simple Transformation Formulas
“Simple” transformation formulas are typically applied to all wells on the plate. For
example:
•
To divide the RU in each well on the plate by 2 and then multiply by 100,
the formula is: (RU/2)*100
•
To raise the RU in each well to the power of 10, the formula is: RUPWR10
Transformation Variable (TVAR)
For more complex transformations, a Transformation Variable (TVAR) can be defined for
use within a transformation formula. This variable defines the scope of the transformation:
whether to apply the transformation to all of the wells on the plate (RU), or to just the
sample wells (SMP).
SCOPE
SMP
•
VARIABLE:
RU
RU
If SMP is chosen:
 The transformation formula will be applied to the sample wells only.
 SMP and any other well identifiers (BLK, PC, NC, STD, etc.) defined will become
available as MAP options when building the transformation formula.
Example:
The assay plate map contains 2 NC wells and 2 PC wells. The remainder of the
map is filled with samples. The assay data reduction requires that the mean of
the NC be subtracted from all the samples on the plate.
The transformation formula is: SMP-NC;x
FLx800 Operator’s Manual
82 | Chapter 4: Operation
•
If RU is chosen, the formula definition screen will appear so that you can define a
formula for use within the transformation formula.
 Use the formula keys (Math, Other, Map and Function) to define the
Transformation Variable (TVAR). Once the variable has been defined, it can be
used in a transformation formula. The TVAR will be available as a MAP option
when building the transformation formula.
Example:
The assay plate map has 2 blanks, 1 control well in duplicate (CTL1), 1 negative
control well in triplicate (NC), and 5 standards in duplicate (STD1-STD5).
The assay data reduction states:
 Subtract the mean of CTL1 from the mean of the NC. Subtract the
difference from all RU’s on the plate.
 Divide the result of the above by the mean of the NC less the mean of
CTL1, then multiply by 100.
On paper, the formula reads:
(RU - (NC;x-CTL1;x)) / (NC;x-CTL1;x) * 100
On the reader, the formula (NC;x-CTL1;x) will be programmed as the TVAR,
since the transformation will apply to all standards, controls and samples on the
plate. To do this:
 At the Scope Variable selection screen, choose RU and press ENTER.
 Enter the formula (NC;x-CTL1;x) using the Math, Other, Map and
Function keys. Press ENTER.
 The formula selection screen is displayed. Choose TRANS.
 Enter the formula (RU-(TVAR))/(TVAR)*100 using the Math, Other, Map
and Function keys. “TVAR” is available as a MAP option.
Example:
In the case of competitive reactions, converting fluorescence/luminescence
data to percent B/B0 can be: (RU/STD1)*100. This divides all the wells by
STD1, presumably the 0 standard, and multiplies the results by 100. To do this:
 At the Scope Variable selection screen, choose RU and press ENTER.
 Enter simply STD1 as the TVAR formula. Press ENTER.
 The formula selection screen is displayed. Choose TRANS.
 Enter the formula (RU/TVAR)*100 using the Math, Other, Map and
Function keys. “TVAR” is available as a MAP option.
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DEFINE |
83
Cutoff Formulas
A cutoff formula calculates a cutoff value that is used for classifying samples. See
Formula Type on page 76 for instructions on selecting an assay and accessing the Cutoff
formula definition screen.
During data reduction, results are evaluated against the cutoff value (with an optional
greyzone), and each well is assigned a call POS (positive), NEG (negative), or EQUIV
(equivocal).
•
One cutoff formula may be defined per assay.
•
If Transformation Formulas are defined, cutoffs are based on the transformed
results. Turn to page 84 to review the results calculation structure.
•
A cutoff formula can consist of a simple numeric value (1.500), a well identifier
(PC, or PC;x to represent the mean), or a formula combining the two (NC;x+0.050).
•
A “greyzone” around the cutoff value can be defined, to indicate equivocal results.
•
Do not use the < or > mathematical symbols in a cutoff formula.
•
Tip: Choose to print a Column Report to see the greyzone and cutoff values, as
well as the equations used to assign calls to samples. See Specifying Data Output
and Reporting Options in Chapter 4 for information on Column Reports.
Greyzone
The greyzone is a definable area around the cutoff value. Samples falling within an area
defined by the greyzone (ex. ± 5% of the cutoff value) could be considered equivocal
(EQUIV).
ENTER
GREYZONE:
10%
•
Use the NUMERIC keys to enter the greyzone percentage.
•
The valid entry range is from 00 to 99%. An entry of 00% indicates no greyzone,
although a sample equal to the cutoff value will still receive the EQUIV call.
•
See POS / NEG Calls on the next page for information on how calls are assigned.
FLx800 Operator’s Manual
84 | Chapter 4: Operation
POS / NEG Calls
After the greyzone is defined, calls for the sample wells (POSitive, NEGative, EQUIVocal),
must be defined.
SAMPLE>CUTOFF+10%:
POS
NEG
POS
•
Select POS or NEG to select the call that will be assigned to samples greater than
the cutoff value plus the greyzone.
•
If for example POS is selected in the screen shown above, calls will be assigned
according to the following equations (SMP represents the sample wells):
EQUIV: SMP <= (CUTOFF+(CUTOFF*GREYZONE)) AND
POS:
NEG:
SMP >= (CUTOFF-(CUTOFF*GREYZONE))
SMP > (CUTOFF+(CUTOFF*GREYZONE))
SMP < (CUTOFF-(CUTOFF*GREYZONE))
Examples
1. The cutoff between negative and positive calls should be calculated as the
average of the negative controls plus the RU value of 5000. Samples greater
than the cutoff should be labeled as positive. No greyzone is required.
•
For this example, NC;x (the mean of the NC wells) equals 20000 RU
•
The cutoff formula is NC;x+5000
•
The greyzone is 00%
•
POS is selected for SAMP > CUTOFF+00%
•
Calls are assigned to sample wells as follows:
 EQUIV if the sample equals 25000
 POS if the sample is greater than 25000
 NEG if the sample is less than 25000
BioTek Instruments, Inc.
DEFINE |
2.
85
For a quantitative assay, samples with RU values greater than the STD2 mean
plus a 10% greyzone should be labeled as positive, samples with RU values less
than the STD2 mean minus the 10% greyzone should be labeled as negative. All
other samples should be considered equivocal.
•
For this example, STD2;x (the mean of the STD2 wells) equals 15000 RU
•
The cutoff formula is simply STD2;x
•
The greyzone is 10%
•
POS is selected for SAMP > CUTOFF+10%
 Calls are assigned to sample wells as follows:
 EQUIV if the sample is greater than or equal to 13500 and
less than or equal to 16500
 POS if the sample is greater than 16500
 NEG if the sample is less than 13500
General Formulas
General Formulas are calculated after all other calculations are complete, and the results
are not used in further data reduction.
 General Formulas are not supported in the current version of the reader.
FLx800 Operator’s Manual
86 | Chapter 4: Operation
Define CURVE
To define curve-fitting parameters for an assay
1.
Start at the Main Menu and select DEFINE.
2.
Select the assay then press ENTER. The DEFINE options screen will appear.
DEFINE:
METHOD MAP
3.
FORMULA
CURVE
Select CURVE. The definable curve-fitting parameters include:
•
Curve-Fit Type
•
Editing of Outliers
•
Axis Identification
•
Extrapolation of Unknowns
DEFINE
METHOD MAP FORMULA CURVE
SELECT CURVEFIT TYPE
EDIT STANDARD
OUTLIERS?
SELECT X/Y
AXIS TYPE
EXTRAPOLATE
UNKNOWNS?
DEFINE
METHOD MAP FORMULA CURVE
Figure 25: Options available under Define CURVE
BioTek Instruments, Inc.
DEFINE |
87
Curve-Fit Type
The FLx800 supports 7 different curve-fitting methods, Linear, Quadratic, Cubic, 4-P, 2-P,
cubic-spline, and point-to-point.
CURVE-FIT TYPE: LINEAR
NONE LINEAR QUAD
*MORE
•
Select *MORE to see additional curve-fit types.
•
NONE: Choose NONE if no standard curve will be generated (this is the
•
•
default).
LINEAR: A simple best-fit straight line is plotted using the values of the
standards.
QUADratic: Uses the Quadratic equation “ax2 +bx +c=y” to plot the standards
values. Utilizing this curve, any data point for a standard that deviates from the
ideal value will not affect the entire curve.
•
CUBIC: Uses the equation “ax3 + bx2 + cx + d = y” to plot the standards values.
•
2-P (Logit/Log): A curve fitted to the standard values, which is
•
C-SPLINE (Cubic-Spline): A piecewise polynomial approximation
This type of curve fit is affected even less than the quadratic fit when any
particular standard has a poor value.
characterized by a skewed sigmoidal (S-shaped) plot that eventually becomes
asymptotic to the upper and lower standard values. The logistic equation is
algebraically transformed to a simpler form in which experimentally
determined values are used for the responses at concentrations of zero and
infinity.
consisting of joining a set of data points by a series of straight lines, which is
then smoothed by using a cubic fit.
•
4-P (4-Parameter Logistic): A curve fitted to the standard values, which is
•
PT-PT (Point-to-Point): A plot that connects each standard point with a line,
characterized by a skewed sigmoidal (S-shaped) plot that eventually becomes
asymptotic to the upper and lower standard values. The 4 parameters are: Left
asymptote, Right asymptote, Slope and Value at the Inflection point. This fit is
most recommended for immunoassay data, and is more exact than Logit/Log.
with no averaging of the values to “smooth” the curve at each standard.
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88 | Chapter 4: Operation
Edit Standard Outliers
After the standard curve has been generated, one or more standards can be excluded from
the recalculation of the curve.
EDIT STD OUTLIERS:MANUAL
NONE MANUAL
•
Select NONE to suppress the Edit Standard Outliers capability for this assay.
•
Choose MANUAL to enable the capability.
 If AVERAGE STANDARDS is set to NO, the individual standard replicates are
available for editing. If set to YES, the standard groups are available for editing.
 After the assay is run and reports are generated, press REPORT from the Main
Menu. Press RESULT, select the assay, and then press ENTER. The EDIT STD
OUTLIERS? YES/NO prompt will appear. See Editing Standard Outliers
later in this chapter for further instructions.
X/Y Axis Type
After the curve-fit type is selected, select the X/Y Axis Type.
X/Y
LIN
AXIS TYPE: LIN
LIN/LOG LOG LOG/LIN
•
Choose the method by which the X and Y axes will be scaled.
•
This option is not available for the 2-P and 4-P curve-fit types. The X/Y scaling
for these curves is always LIN/LIN.
Extrapolation of Unknowns
The FLx800 provides the option to “extrapolate” the curve to evaluate samples outside of
the fluorescence/luminescence range defined by the standards.
EXTRAPOLATE
YES
NO
UNKNOWNS?YES
•
Select YES to enable Extrapolation, otherwise select NO.
•
On printed reports, extrapolated concentrations (RSLT values) are surrounded by
< >, for example <44.425>.
BioTek Instruments, Inc.
DEFINE |

89
If extrapolation is chosen for the Point-to-Point curve fit,
unknown concentrations will be extrapolated linearly from the
nearest segment of the curve. If the plot includes both increasing
and decreasing segments, the curve printout will be labeled
“Ambiguous.” The resulting values, which actually are
extrapolated, may not be indicated as such. All calculated results
for an “Ambiguous” curve should be considered unreliable.
Panel Assays
A Panel assay is a collection of up to 8 assays to be run on one plate.
•
Only one panel can be defined on the reader at any time.
•
The assays specified within the Panel must be pre-defined in any of the assay
positions 1-55.
•
The assays specified within the panel must all use the Endpoint read method and
read at the same wavelength(s).
•
Any curve-fit type, formulas or standard concentrations previously defined for
each assay will be used when the assay is selected for a Panel.
•
Panel assays cannot re-use standard curves.
•
The type and number of controls, blanks, standards and replicates in the assays
chosen for the Panel will be “copied” into the Panel definition. To change any of
the map or assay parameters in the Panel, they must be changed in the pre-defined
assay first.
•
Considering printing a Map Report for each assay that will be included in the
panel, for use with mapping the Panel.
To create a panel assay:
1.
Start at the Main Menu, select DEFINE, then choose assay number 99. Enter
the panel assay name.
NAME:
-
PANEL
/
:
SPACE
2.
The default name is “PANEL”. Use the ALPHA and NUMERIC keys to update
the Assay name, if desired.
3.
Press ENTER to continue. The Number of Assays entry screen will appear.
NUMBER
4.
OF
ASSAYS:
2
Specify the number of assays to include in the panel (1 to 8).
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90 | Chapter 4: Operation
5.
Press ENTER to continue. The Mapping Direction selection screen will appear.
This option ensures that all assays will be mapped in the same direction.
MAPPING DIRECTION:DOWN
DOWN
ACROSS
6.
Select DOWN or ACROSS.

7.
The original mapping directions for the pre-defined assays are
overridden by the Panel’s mapping direction. If the assays
include replicates, they will follow the Panel mapping direction.
After selecting the mapping direction of the assays, choose which assays to
include in the panel. Press OPTION to cycle through the assay numbers and
names, or use the NUMERIC keys to enter an assay number. Press ENTER to
make a selection.
SELECT ASSAY NUMBER:
NAME: DNA QUANT
8.
After selecting an assay, its starting location must be defined. Use the ALPHA
and NUMERIC keys to choose the well location to begin the assay. Wells A01
through H01 are valid for ACROSS mapping; A01 through A12 are valid for
DOWN mapping.
START MAPPING
AT WELL LOCATION:
9.
22
A01
Repeat this process for each assay within the panel. Remain aware of the total
number of controls, standards, and blanks that were originally mapped in each
individual assay while mapping for the Panel assay.
For example, to include Assays 1, 8 and 22 in the Panel assay (DOWN mapping
is selected for the Panel):
•
Assay 1 has a total of 12 wells defined for controls, blanks, and
standards. In the Panel, the mapping for Assay 1 begins in well A01.
The user wants to run 6 samples in Assay 1. Assay 1 now fills wells A01
through B03.
•
The mapping for Assay 8 can begin in well B04, or any well other than
A01 to B03. The reader will “beep” if you try to map into a well that is
already assigned for use with the Panel.
•
The mapping for Assay 22 may begin at the next available well location
after Assay 8 mapping is complete.
BioTek Instruments, Inc.
READ |
91
•
After all the assays have been entered into the Panel, consider printing
the Panel’s Map Report to verify the map before reading the plate.
Choose Report (from the Main Menu), Map, Assay 99. The reader will
print the map of each assay configured in the Panel.
•
The Panel Assay results are sorted by Sample (unless a custom assay has
been programmed by BioTek).
 The Interpretation of Results reports for each assay in the panel will print
first, and then the Sample results will print.
READ
The Main Menu option READ allows you to select an assay to run, define any required run-time
options, then begin a plate read.
 Before reading a plate, make sure the reporting options are set correctly
under UTIL > OUTPUT. See Specifying Data Output and Reporting
Options in Chapter 4 for more information.
To read a plate:
1.
Start at the Main Menu and select READ.
2.
The Select Assay Number screen will appear.
3.
Select an assay, then press ENTER. See Selecting an Assay to Run on the next page
for detailed instructions.
4.
If required, enter the Number of Samples, Plate ID, and/or Sample ID.
 The options to present these screens are configurable by selecting UTIL >
READ. See Selecting Read Options in Chapter 4 for more information.
ENTER NUMBER
SAMPLES:
PLATE
ENTER
SAMPLE
5.
ID:
/
ID:
OF
1234A
:
20
SPACE
0001
Place the plate in the carrier, then press the READ key to continue.
FLx800 Operator’s Manual
92 | Chapter 4: Operation
PLACE PLATE IN CARRIER
AND PRESS <READ> KEY
Selecting an Assay to Run
To select an assay to run, start at the Main Menu and select READ. The Select Assay Number
screen will appear:
SELECT ASSAY NUMBER:01
NAME:DNA QUANT
•
Use the numeric keys to enter the number of any predefined assay stored in the
reader’s memory, or the Option key to advance one assay at a time.
•
Press ENTER to select the assay and continue.
Run-Time Prompts
After the assay is selected, one or more informational prompts may be presented, depending
on preferences selected via UTIL > READ, whether or not the assay specifies manual
mapping, or if this is a custom pre-programmed assay.
•
Prompts enabled via UTIL > READ can include Enter Number of Samples, Plate ID,
and Enter Sample ID.
•
If the assay specifies manual mapping, prompts for information will include the
locations for the sample wells.
•
If running a custom assay, typical prompts might include:
 The number of samples
 Standard concentrations
 Assay ID
 Fill pattern
 Blank method
 First well location
 Replicate count for each well type
 Excitation and/or Emission filter
 Report preferences, etc.
Enter Number of Samples
If the Enter Number of Samples prompt is presented, indicate the number of sample groups
on the plate. The number of sample replicates is typically pre-defined in the assay, but if
this is a custom assay, you may also be prompted to enter the replicate count.
BioTek Instruments, Inc.
READ |
ENTER
NUMBER
OF
SAMPLES:
93
20
•
Use the numeric keys to enter the number of sample groups.
•
The valid entry range is from 01 to the maximum number of wells remaining on
the plate after any blank, control, or standard wells are mapped.
•
If you enter a value greater than the number of empty wells remaining on the
plate, the reader will “beep” and automatically change the value to the maximum
permissible number of samples.
Enter Plate ID
If the Plate ID prompt is presented, enter a unique plate identifier to be stored in memory
with the assay name and fluorescence/luminescence data.
PLATE
-
ID:
/
1234-A
:
SPACE
•
Use up to 10 alphanumeric characters. See page 48 for instructions on using the
keypad.
•
Although the reader does not require it, consider entering a unique ID for each
plate, especially if reading multiple plates for the same assay.

Use caution when creating multiple Plate IDs. The reader does
not provide a warning when the maximum of 8 Plate IDs stored
in memory is about to be exceeded. If a 9th Plate ID is added, it
will overwrite the first Plate ID stored in memory.
Enter Sample ID
If the Enter Sample ID prompt is presented, enter a starting sample identification number.
ENTER
SAMPLE
ID:
0001
•
The valid entry range is from 0001 to 9999.
•
The software will automatically increment each subsequent sample
identification by 1.
•
The sample IDs will be assigned in accordance with the plate map defined in
the assay.
FLx800 Operator’s Manual
94 | Chapter 4: Operation
Enter Well Location
If the assay specifies manual plate mapping and if Prompt for Sample Count is set to Yes
under UTIL > READ, sample well locations can be defined at run-time.
SAMPLE#
1
LOCATION
REP# 1:A02
•
The sample well locations originally defined in the assay will be presented.
If desired, use the keypad to enter new well location for each sample replicate.
•
Press ENTER to advance to the next replicate.
Beginning the Plate Read
When all required prompts have been responded to, the Place Plate in Carrier prompt
appears.
PLACE PLATE IN CARRIER
AND PRESS <READ> KEY
•
Before reading the plate, make sure that the printer is connected, turned on, and
full of paper.
•
Place the plate on the carrier, then press the READ key to initiate the plate read.
•
After the read is complete, data reduction will perform (“Calculating Results…”),
and then the reports will be generated (“Generating Reports…”).
•
To halt the read in progress, press the STOP key.

If using the incubation option, the reader will wait for the
incubator to reach temperature before reading the plate.
BioTek Instruments, Inc.
REPORT |
95
REPORT
Reports are automatically generated after a plate has been read (see Specifying Data Output
and Reporting Options in Chapter 4 for information on selecting reports). Results reports
also can be regenerated manually by using the REPORT option from the Main Menu. In
addition, Map, Assay, and Assay List reports can be printed.
 See Appendix C for sample reports.
READY
READ
09:45AM
0 9 / 1 7 / 10
DEFINE REPORT UTIL
PRINT REPORT?
RESULT
MAP ASSAY
•
LIST
Select RESULT to obtain an exact copy of results from the plate reading (the 8 most
recent sets of plate data are stored in memory).
 The form in which the results are presented is determined by the report settings
(Matrix, Column, Curve Fit) specified under UTIL > OUTPUT.
•
Select MAP to print a matrix showing the locations of the Blanks, Standards,
Controls and Samples for a particular assay.
•
Select ASSAY to print a plate map and a listing of all of an assay’s settings, such as
wavelengths, numbers of well types, formulas, and curve fit parameters.
•
Select LIST to print a list of all assays (name and number), currently programmed
in the reader.
Results Report
The reader stores the data for the 8 most recent plate reads. Results reports can be
generated for these plates if, for example, the data that automatically printed after the read
needs to be printed in a different format, or if the standard curve contains outliers that
require editing.
REPORT:SSDNA
ID: 0001
10/11/10
•
The most recently read plate is presented first, showing the assay name, the plate
ID (if one was entered), and the date the plate was read.
•
Press OPTION to see the next plate in memory.
•
Press ENTER to select a plate and continue.
FLx800 Operator’s Manual
96 | Chapter 4: Operation
•
If a standard curve was generated and EDIT STANDARD OUTLIERS was set to
Manual in the assay definition, the Edit Std Outliers? prompt is presented,
otherwise, the Print Results? prompt is presented.
Editing Standard Outliers
If a standard curve is generated and if EDIT STANDARD OUTLIERS is set to MANUAL
in the assay definition, the option to edit outliers is presented.
EDIT
YES
STD
NO
OUTLIERS?
•
Select NO to include all standards in the curve fit calculations.
•
Select YES to indicate that one or more standard replicates or groups should be
temporarily excluded from curve fit calculations.
 If AVERAGE STANDARDS was set to NO in the assay definition, one or more
standard replicates can be chosen for exclusion.
EDIT
YES
STD1
NO
REP1?
YES
•
Select YES to exclude the replicate from curve fit calculations.
•
Select NO to retain the replicate.
•
Press ENTER to advance to the next replicate.
•
If AVERAGE STANDARDS was set to YES in the assay definition, one or
more standard groups can be chosen for exclusion.
EDIT
YES
STD1;X?
NO
YES
•
Select YES to exclude the group from curve fit calculations.
•
Select NO to retain the group.
•
Press ENTER to advance to the next group.

Each curve-fit type requires a minimum number of standards for
curve generation: 4 for 2-P, 4-P, cubic, and cubic-spline, 3 for
quadratic, and 2 for linear and point-to-point. Exercise caution
when editing outliers. If the assay is left with insufficient
standards, the curve fit will fail.
Printing Results
After the assay is selected and standard outliers are edited (if necessary), the results report
can be printed.
BioTek Instruments, Inc.
REPORT |
PRINT
YES
97
RESULTS?
NO
•
Make sure that the printer is connected, turned on, and full of paper.
•
Select YES to print reports, or NO to return to the Main Menu.
Map Report
The Map Report contains a matrix in Row x Column format, showing the location of
every well identifier defined in the plate map.
SELECT
NAME:
ASSAY
SSDNA
NUMBER:
03
•
Press OPTION to cycle through the list of available assays, or enter the number of
the desired assay.
•
Press ENTER to print the report.
Assay Report
The Assay Report lists the assay definition parameters and their current settings.
SELECT
NAME:
ASSAY
SSDNA
NUMBER:
03
•
Press OPTION to cycle through the list of available assays, or enter the number
of the desired assay.
•
Press ENTER to print the report.
List Report
The List Report lists the all of the assays (name and number), currently programmed on
the reader.
•
Select REPORT from the Main Menu, and then select LIST to print the report.
FLx800 Operator’s Manual
98 | Chapter 4: Operation
UTILITY
The UTILITY option allows you to set up the date and time, specify your data output and
report options and select your read options.
Setting the Date and Time
To set the current Date and Time, and/or to change their formats:
1.
From the Main Menu, select UTIL > SETUP. The Edit Setup Information menu will
appear.
EDIT SETUP INFORMATION
DATE
TIME FILTER *MORE
2.
Select DATE. The DATE entry screen will appear.
DATE:
MMDDYY
3.
1 1 / 1 7 / 10
DDMMYY
MDY
•
Enter the date using the numeric keys on the keypad. The cursor is positioned
under the first editable field, and advances automatically.
•
To change the date format, select MMDDYY or DDMMYY. The display
automatically updates to reflect the new format.
•
Press ENTER to return to the Edit Setup Information menu.
To change the current time and/or the time format, select TIME from the Edit Setup
Information menu. The TIME entry screen will appear.
TIME:
12HOUR
03:12PM
24HOUR
12HOUR
AM/PM
•
Enter the time using the numeric keys on the keypad. The cursor is positioned
under the first editable field, and advances automatically.
•
To change the time format, select 12HOUR or 24HOUR, then AM or PM. The
display automatically updates to reflect the new format.
•
Press ENTER to return to the Select Utility Options menu.
BioTek Instruments, Inc.
UTILITY |
99
Specifying Data Output and Reporting Options
 These options have no effect if the instrument is being controlled by Gen5.
Plate data can be sent to an attached printer or external computer.
•
The on-board software provides several different options for report format and
content.
•
Data sent to an external computer has no data reduction applied to it, with the
exception of dual-wavelength subtraction (if defined in the assay). Any reporting
options selected through the on-board software have no effect on serial output.
To specify data output and reporting options, start at the Main Menu and select UTIL >
SETUP > OUTPUT. The Report Output screen will appear.
REPORT
PRINT
OUTPUT? PRINT
COMPUTER
BOTH
•
The current output option is displayed on the top line. Select PRINT to send
reports directly to a printer, COMPUTER to send data out through the serial port,
or BOTH.
•
Press ENTER to continue. The Select Printer screen will appear.
SELECT
EPSON
PRINTER:
HP
EPSON
 The FLx800 supports printers using either HP’s PCL3 language, such as the HP
DeskJet series, or Epson’s LQ language. For the latest list of compatible printers
contact BioTek Instruments’ Technical Assistance Center (see Appendix B for
details).
 Select EPSON or HP as appropriate.
 Press ENTER to continue. The Report Type screen will appear.
REPORT
COLUMN
TYPE?
MATRIX
MATRIX
BOTH
 The currently selected report type is displayed in the top line. Select COLUMN to
print information in a list (columnar) format, MATRIX to print in a format that
resembles the plate type (ex. 8 x 12 matrix), or BOTH.
 Appendix C contains sample Column and Matrix Reports.
 Press ENTER to continue. The Samples in Col Rpt screen will appear.
SAMPLES
YES
FLx800 Operator’s Manual
IN
NO
COL
RPT?
NO
100 | Chapter 4: Operation
 Select YES to print results for all wells on the plate, including samples.
 Select NO to limit the results information to blanks, controls, and standards.
 Press ENTER to continue. The Print Curve-Fit screen will appear.
PRINT
YES
CURVE-FIT?
NO
YES
Select YES to print the standard curve (if the assay generates one), or NO to
suppress this report.
 Press ENTER to return to the Select Utility Option menu.

Selecting Read Options
At plate-read time, the software can be configured to present the user with a series of
prompts, to enter information such as Plate ID, Sample ID, and/or Sample Count.
To specify various read-time options, start at the Main Menu and select UTIL > READ.
 The Prompt for Plate ID screen will appear. Press ENTER to cycle through the
prompt screens.
PROMPT
YES
FOR
NO
PLATE
PROMPT
YES
FOR
NO
SAMPLE
PROMPT
YES
SAMPLE
NO
ID?
ID?
COUNT?
YES
NO
YES
 If selected, at read-time:
PLATE ID prompts for microplate identification.
SAMPLE ID prompts for identification for each sample group.
SAMPLE COUNT prompts for the number of samples on the plate.
 Pressing ENTER after each selection advances the display.
 When selections are completed, the display returns to the Select Utility Option
menu.
BioTek Instruments, Inc.
Chapter 5
Instrument Qualification
This chapter contains procedures for qualifying the initial and ongoing
performance of the FLx800 and the external dispenser module (if
used).
Overview................................................................................. 102
IQ/OQ/PQ ............................................................................... 102
Recommended Qualification Schedule ......................................... 104
System and Checksum Tests ..................................................... 105
Corners, Sensitivity, and Linearity Tests ...................................... 107
Required Materials............................................................... 108
Solution Preparation ............................................................ 109
Procedure – Gen5 ............................................................... 110
Procedure – Keypad ............................................................ 111
Pipette Map for Sodium Fluorescein Tests ............................... 112
Results Analysis .................................................................. 113
Troubleshooting .................................................................. 114
Gen5 Protocol Reading Parameters ........................................ 115
Onboard Assay Parameters................................................... 116
Using Methylumbelliferone.................................................... 117
Dispense Accuracy & Precision Tests ........................................... 123
Required Materials............................................................... 123
Test Solutions ..................................................................... 124
Test Setup ......................................................................... 125
Procedure........................................................................... 125
Results Analysis .................................................................. 127
Gen5 Test Protocols ............................................................. 128
102 | Chapter 5: Instrument Qualification
Overview
Every FLx800 instrument and external dispenser module is fully tested at BioTek prior to
shipment and should operate properly upon initial setup. If you suspect that a problem
occurred during shipment, if you have received the equipment after returning it to the factory
for service, and/or if regulatory requirements dictate that you qualify the equipment on a
routine basis, you should perform the procedures outlined in this chapter.
This chapter contains BioTek Instruments’ recommended Installation Qualification (IQ),
Operational Qualification (OQ), and Performance Qualification (PQ) procedures for all models
of the FLx800 Multi-Detection Microplate Reader.

A Product Qualification Package (PN 7080532) for the FLx800
is available for purchase. The package contains complete
procedures for performing Installation Qualification, Operational
Qualification, Performance Qualification, and Preventive
Maintenance. Microsoft® Excel spreadsheets are provided for
performing the calculations, and checklists and logbooks are
provided for recording results. Contact your local BioTek dealer for
more information.
IQ/OQ/PQ
Installation Qualification confirms that the reader and its components have been supplied
as ordered, and ensures that they are assembled and configured properly for your lab
environment.
•
The recommended IQ procedure consists of setting up the instrument and its
components as described in Chapter 3, Installation and performing the System and
Checksum Tests.
•
The IQ procedure should be performed initially (before the reader is used for the first
time).
•
The successful completion of the IQ procedure verifies that the instrument is installed
correctly. The Operational Qualification procedure should be performed immediately
following the successful IQ.
Operational Qualification confirms that the equipment operates according to specification
initially and over time.
•
The recommended OQ procedure consists of performing the System Test, Checksum
Test, Corners Test, and Sensitivity Test. If the external dispenser module is used, the
BioTek Instruments, Inc.
IQ/OQ/PQ |
103
recommended OQ procedure also consists of performing the Dispense Accuracy and
Precision Test.
•
The OQ procedure should be performed initially (before first use) and then routinely;
the recommended interval is annually. It should also be performed after any major
repair or upgrade to the hardware or software.
•
Although out-of-tolerance failures will be detected by the OQ tests, results should be
compared with those from the routine Performance Qualification tests and previous
OQ tests to monitor for trends.
•
The successful completion of the OQ procedure, in combination with results that are
comparable to previous PQ and OQ tests, confirms that the equipment is operating
according to specification initially and over time.
Performance Qualification confirms that the equipment consistently meets the
requirements of the tests performed at your laboratory.
•
The recommended PQ procedure consists of performing the System Test, Checksum
Test, Corners Test, and Sensitivity Test. If the external dispenser module is used, the
recommended PQ procedure also consists of performing the Dispense Accuracy and
Precision Test.
•
Your facility’s operating policies may also require that you routinely perform an actual
assay as a part of the PQ, to confirm that the equipment will consistently give
adequate results for the assays to be run with it.
•
These tests should be performed routinely; the recommended interval is monthly or
quarterly, depending on the test. This frequency may be adjusted depending on the
trends observed over time.
•
The successful completion of the PQ procedure confirms that the equipment is
performing consistently under normal operating conditions.
FLx800 Operator’s Manual
104 | Chapter 5: Instrument Qualification
Recommended Qualification Schedule
The following schedule defines the factory-recommended intervals for qualifying an FLx800
used two to five days a week. The schedule assumes the reader is properly maintained as
outlined in Chapter 6, Preventive Maintenance.
Important: The risk factors associated with your assays may require that the Operational and
Performance Qualification procedures be performed more frequently than shown below.
Recommended Instrument Qualification Schedule for the FLx800
Installation
Qualification
Operational
Qualification
Performance
Qualification
Initially
Initially &
Annually
Monthly



Corners Test


Sensitivity Test


Linearity Test


Dispense Accuracy and
Precision Test

Tests
System and Checksum Tests
Quarterly

•
The System Test checks voltage levels, motor control, and the integrity of the
photomultiplier. See page 105.
•
The Checksum Test compares the on-board software with internally recorded
checksum values to ensure that no corruption has occurred. See page 107.
•
The Corners Test uses fluorescent compounds to verify that the plate carrier is
properly aligned in relation to the optical probe(s). See page 107.
•
The Sensitivity Test uses fluorescent compounds to verify that the difference
between the means of wells with known lower limits of concentration of the substance
under investigation is statistically distinguishable from the mean of wells with pure
diluent. See page 107.
•
The Linearity Test verifies that the system is linear; that is, signal changes
proportionally with changes in concentration. Proving that the system is linear allows
the Sensitivity Test to be run on two points instead of using serial dilutions. See page
107.
•
The Dispense Accuracy and Precision Test checks that the dispense volumes are
within the tolerance specification for the instrument. This test only applies to models
with the external dispenser module. See page 123.
BioTek Instruments, Inc.
System and Checksum Tests |
105
System and Checksum Tests
The System and Checksum Tests are performed automatically whenever the instrument is
turned on. They can also be performed manually through the FLx800 Main Menu or via Gen5.
•
The System Test begins with a check of the stepper motors and the analog power
supplies, to ensure that they have a proper input voltage level. The data flash
checksum, motor axis, and analog offset are then verified. PMT dark current, noise,
and gain are checked to ensure they fall within specific pass/fail criteria. If equipped,
incubation zones are measured and reported.
•
The Checksum Test compares the on-board software with internally recorded
checksum values to ensure that no corruption has occurred. If this test is run
manually, part number and version information is displayed for the software currently
loaded on the instrument. This information is useful when contacting BioTek for
technical assistance. See Appendix B, Troubleshooting for more information.
When the System and Checksum Tests are performed, the test results are shown on the
display. If the tests pass, SYSTEM TEST PASS appears briefly. If there is a failure, the
instrument “beeps” and displays an error code. See Appendix B, Error Codes for more
information. Additional information is printed (System Test) or displayed (Checksum Test) if
the tests are run manually.
If the FLx800 is running in standalone mode:
•
From the Main Menu, select UTIL > TESTS > SYSTEM to run the System Test, or
CHKSUM to run the Checksum Test.
 If the System Test is run, the System Self Test Report will print, in a pass/fail
format.
 If the Checksum Test is run, the information displayed will resemble the
following:
7080201
VERSION 1.10
CODE CHECKSUM:
(085D)
•
The initial checksum test display will show the on-board (base code)
software part number, version number, and checksum.
•
After a few moments, a second screen will display showing the assay
configuration software part number and version number:
7080210-FW
FLx800 Operator’s Manual
V1.00.00
106 | Chapter 5: Instrument Qualification
If the FLx800 is controlled by Gen5:
•
If the reader is equipped with an incubator and you have not already turned it on,
access the System menu and select Reader Control. Click the Pre-Heating tab. Enter
a Requested temperature of at least 37°C and then click On. Wait until the
temperature on the display reaches the set point before continuing.
•
From the Gen5 main screen, select System > Diagnostics > Run System Test.
•
When the test is complete, a dialog will appear to request additional information.
Enter the information (if desired) and click OK.
•
The results report will appear. Scroll down toward the bottom; the text should read
SYSTEM TEST PASS. Print the report and store it with your Installation records.
11:05AM 12/01/05
SYSTEM SELF TEST
7080207 Version 1.15
181039
Bias current offset
Offset voltage
750V measurement
750V noise
500V measurement
500V noise
-0.4
1620
7.7
14
1.5
1
counts
counts
counts
counts
counts
counts
1100
PASS
PASS
PASS
INCUBATOR SELF TEST
Temperature Setpoint:
Zone 1: 36.9
Zone 2: 37.0
Zone 3: 37.1
Door: CLOSED
37.0
Min: 36.9
Min: 36.9
Min: 36.9
Max: 36.9
Max: 37.0
Max: 37.1
Upper
Lower
Lower
Upper
Delta
Delta
Delta
Delta
PROBE:
Upper
Lower
Lower
Upper
Delta
Delta
Delta
Delta
TOP
Left Corner: x=
Left Corner: x=
Right Corner: x=
Right Corner: x=
1: 9724 - 9716=
2: 1036 - 1032=
3:
268 - 268=
4: 5792 - 5796=
Range: PASS
Range: PASS
Range: PASS
A/D Test: PASS
Thermistor: PASS
Thermistor: PASS
Thermistor: PASS
The INCUBATOR SELF TEST shows the PASS or FAIL
condition of the heating zones in the incubator (if
equipped). See Incubator Self-Test on the next page.
AUTOCAL ANALYSIS
PROBE:
Current Average: 37.0
9724
9716
1032
1036
8
4
0
-4
y= 268
y= 5796
y= 5792
y= 268
9720
9712
1032
1032
8
0
0
4
y=
y=
y=
y=
AUTOCAL ANALYSIS provides coordinates of
the last Autocal performed on the instrument.
AUTOCAL verification is not included in the
SYSTEM TEST result given below.
BOTTOM
Left Corner: x=
Left Corner: x=
Right Corner: x=
Right Corner: x=
1: 9720 - 9712=
2: 1032 - 1032=
3: 1848 - 1848=
4: 7380 - 7376=
1848
7376
7380
1848
The SYSTEM TEST result will be either PASS or
FAIL. If it fails, an error code is included.
SYSTEM TEST PASS
Figure 26: Sample System Test Report (the format varies depending on how the test is initiated).
BioTek Instruments, Inc.
Corners, Sensitivity, and Linearity Tests |
107
Corners, Sensitivity, and Linearity Tests
The Corners, Sensitivity, and Linearity Tests are performed on every FLx800 before it leaves the
factory. These tests are critical for verifying instrument performance.
•
The Corners Test uses fluorescent compounds to verify that the plate carrier is
properly aligned in relation to the optical probe. If your FLx800 is equipped with both
top and bottom probes, we recommend running the test for both probes.
•
The Sensitivity Test uses fluorescent compounds of varying concentrations to test
the fluorescence reading capability of the reader. The ability to detect specific
compounds at low concentrations ensures that the filters, optical path, and PMT are all
in working order. This test verifies that the difference between the means of wells with
known lower limits of concentration of the substance under investigation is
statistically distinguishable from the mean of wells with pure diluent.
•
The Linearity Test verifies that the system is linear; that is, signal changes
proportionally with changes in concentration. Proving that the system is linear allows
the Sensitivity Test to be run on two points instead of using serial dilutions.

Important! The tests presented in this section require specific
microplates, solutions, and EX/EM filters. Your laboratory may
require a deviation from some of these tests. For example, you
may wish to use a different fluorescing solution and/or
microplate.
If deviation from the tests as presented in this section is required,
the following steps should be taken the first time each test is run
(e.g., during the Initial OQ):
FLx800 Operator’s Manual
1.
Perform the tests exactly as described on the following pages.
2.
Re-run the tests using your particular solutions, filters,
microplates, etc. If results are comparable, then the results from
these tests will be your baseline for future tests.
3.
Be sure to document your new test procedure(s), and save all
test results.
108 | Chapter 5: Instrument Qualification
Required Materials
 BioTek offers a liquid test kit (PN 7160013) containing the microplates and
solutions used in this procedure.
 Microplates should be perfectly clean and free of dust or bottom scratches.
Use new microplates from sealed packages.
 FLx800 IBEP model: To effectively read microplates using the top
probe, a plate carrier adapter is required (BioTek PN 7080522)
to raise the microplate and bring it closer to the probe. Microplates with
heights up to 0.600" can be read using this adapter.
 Methylumbelliferone can be used as an alternative or supplemental
method for performing these tests for the top probe. See the instructions
starting on page 117.
•
Buffer Solution:
 NIST-Traceable Sodium Borate Reference Standard (pH 9.18), (e.g., FisherScientific 1 L Sodium Borate Mfr. #159532, or equivalent) or
 Phosphate-Buffered Saline (PBS), pH 7.2-7.6 (Sigma® tablets, Mfr. #P4417, or
equivalent) and pH meter or pH indicator strips with pH range 4 to 10
•
Sodium Fluorescein Powder (1 mg vial, BioTek PN 98155)
•
Bottom optics: A clean Hellma® Quartz 96-well titration plate (Mfr. #730.009.QG),
or equivalent, such as the 96-well glass-bottom Greiner SensoPlate™ (Mfr.
#655892).
•
Top optics: A new, clean, 96-well solid black microplate, such as Corning® Costar
Mfr. #3915.
•
Excitation filter 485/20 nm installed
•
Emission filter 528/20 nm installed
•
Plate carrier adapter (BioTek PN 7080522) for top probe reading (FLx800 TBEP
only)
•
Deionized or distilled water
•
Various beakers, graduated cylinders, and pipettes
•
pH meter or pH indicator strips with pH range 4 to 10
•
95% Ethanol (for cleaning the bottoms of the plates)
•
Aluminum foil
•
(Optional, but recommended) 0.45 micron filter
•
(Optional) Black polyethylene bag(s) to temporarily store plate(s)
BioTek Instruments, Inc.
Corners, Sensitivity, and Linearity Tests |

109
If using BioTek’s sodium fluorescein powder (PN 98155) be
sure to hold the vial upright and open it carefully; the material
may be concentrated at the top.
When diluting the sodium fluorescein powder in PBS, it takes
time for the powder to completely dissolve. Allow the solution to
dissolve for 4 to 5 minutes, with intermittent vortexing, before
preparing the dyes.
Wrap the vial containing the SF stock solution in foil to prevent
exposure to light. Discard the unused solution after seven days.
Discard any open, unused buffer solution after seven days.
Solution Preparation
1.
2.
The Sodium Borate solution does not require further preparation; proceed to step 2. If
you are using PBS, prepare the solution now:
•
(Optional, but recommended) Using a 0.45-micron filter, filter 200 ml of deionized
or distilled water.
•
Follow the manufacturer’s instructions on the PBS packaging to create 200 ml,
dissolving the necessary amount of PBS into the filtered water.
•
Place on a stir table until the PBS is completely dissolved.
•
Check the pH with either a pH meter or with pH indicator strips. The pH should
be between 7.2 and 7.6 at 25°C.
Prepare the sodium fluorescein (SF) stock solution:
Add 2.0 ml of the PBS solution to the 1 mg Sodium Fluorescein (SF) vial. This yields a
1.3288 mM stock solution. Ensure that the dye has completely dissolved and is well
mixed. Ensure that the dye has completely dissolved and is well mixed.
3.
Carefully prepare the dilutions. Label each with “SF” and the concentration:
Mix this SF solution:
with buffer:
to make:
0.53 mL of 1.3288 mM stock solution
13.47 mL
50.2 μM
110 µL of 50.2 μM solution
13.89 mL
400 nM
3.5 mL of 400 nM solution
10.5 mL
100 nM
0.46 mL of 100 nM solution
13.54 mL
3.3 nM
4.24 mL of 3.3 nM solution
9.76 mL
1 nM
FLx800 Operator’s Manual
Corners Test
Sensitivity/Linearity Tests
110 | Chapter 5: Instrument Qualification
Procedure – Gen5
1.
If you have not already done so, prepare the test solutions; see page 109.
2.
If you have not already done so, create Gen5 protocols ‘FLx800 FI_B’ and ‘FLx800 FI_T’
described on page 115.
3.
If applicable, perform the tests using the Bottom optics:
•
4.
Create an experiment based on FLx800 FI_B and read the plate.
•
5.
6.
Pipette the solutions for the Corners, Sensitivity, and Linearity Tests into a clean
96-well quartz or glass-bottom microplate (see the map on page 112).
When complete, save the experiment using a unique name.
If applicable, perform the tests using the Top optics:
•
Adjust the probe height for the plate being used; the probe should be lowered as
close to the plate as possible without interfering with its movement. See Top
Optical Probe Adjustment in Chapter 2.
•
Pipette the solutions for the Corners, Sensitivity, and Linearity Tests into a new 96well solid black microplate (see the map on page 112).
•
Create an experiment based on FLx800 FI_T and read the plate.
•
When complete, save the experiment using a unique name.
When finished, analyze the results using the calculations on page 113.
BioTek Instruments, Inc.
Corners, Sensitivity, and Linearity Tests |
111
Procedure – Keypad
1.
Make sure the printer is attached to the reader and turned on. Configure the reader to
print results in the Matrix Report format.
2.
If you have not already done so, create the ‘FLBOT’ (for Bottom optics) and/or ‘FLTOP’
(for Top optics) assays described on page 116.
3.
If you have not already done so, prepare the test solutions; see page 109.
4.
If applicable, perform the tests using the Bottom optics:
5.
6.
•
Pipette the solutions for the Corners, Sensitivity, and Linearity Tests into a clean
96-well quartz or glass-bottom microplate (see the map on page 112).
•
Read the plate using the ‘FLBOT’ assay. When prompted, enter 96 for the number
of samples.
•
When finished, print the results.
If applicable, perform the tests using the Top optics:
•
Adjust the probe height for the plate being used; the probe should be lowered as
close to the plate as possible without interfering with its movement. See Top
Optical Probe Adjustment in Chapter 2.
•
Pipette the solutions for the Corners, Sensitivity, and Linearity Tests into a new 96well solid black microplate (see the map on page 112).
•
Read the plate using the ‘FLTOP’ assay. When prompted, enter 96 for the number
of samples.
•
When finished, print the results.
Analyze the results using the calculations on page 113.
FLx800 Operator’s Manual
112 | Chapter 5: Instrument Qualification
Pipette Map for Sodium Fluorescein Tests
 Seal the plates with foil or store them in black polyethylene bags until use. For
bottom optics tests, if the base of a plate is touched, clean the entire base with
alcohol (95% ethanol) and then wipe with a lint-free cloth. Before placing a plate
in the instrument, blow the bottom of the plate with an aerosol duster.
For the Corners test:
•
Pipette 200 µL of the 3.3 nM SF solution into wells A1-A3, A10-A12,
H1-H3, and H10-H12.
•
(Optional for plates with black sidewalls) Pipette 200 µL of buffer in the wells
surrounding the 3.3 nM wells (‘CBUF’ in the grid below).
For the Sensitivity/Linearity tests:
•
Use a multi-channel pipette with just four tips installed.
•
Pipette 150 µL of buffer into wells C2-F5 and C10-F12.
•
Pipette 150 µL of the 1 nM SF solution into wells C1-F1. Discard the tips.
•
Pipette 150 µL of the 1 nM SF solution into wells C2-F2.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from wells C2-F2 and dispense into wells C3-F3.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from wells C3-F3 and dispense into wells C4-F4.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from wells C4-F4 and dispense into wells C5-F5.
Mix the wells using the pipette.
•
Aspirate 150 µL from wells C5-F5 and discard.
1
2
3
4
A
3.3
nM
3.3
nM
3.3
nM
B
CBUF
CBUF
C
1.0
nM
D
5
6
7
8
9
10
11
12
CBUF
CBUF
3.3
nM
3.3
nM
3.3
nM
CBUF
CBUF
CBUF
CBUF
CBUF
CBUF
0.5
nM
0.25
nM
0.125
nM
0.0625
nM
BUF
BUF
BUF
1.0
nM
0.5
nM
0.25
nM
0.125
nM
0.0625
nM
BUF
BUF
BUF
E
1.0
nM
0.5
nM
0.25
nM
0.125
nM
0.0625
nM
BUF
BUF
BUF
F
1.0
nM
0.5
nM
0.25
nM
0.125
nM
0.0625
nM
BUF
BUF
BUF
G
CBUF
CBUF
CBUF
CBUF
CBUF
CBUF
CBUF
CBUF
H
3.3
nM
3.3
nM
3.3
nM
CBUF
CBUF
3.3
nM
3.3
nM
3.3
nM
BioTek Instruments, Inc.
Corners, Sensitivity, and Linearity Tests |
113
Results Analysis
Corners Test
1.
Calculate the Mean of the twelve wells containing the 3.3 nM SF test solution
(A1-A3, A10-A12, H1-H3, H10-H12).
2.
Calculate the Standard Deviation of the same twelve wells.
3.
Calculate the % CV: (Standard Deviation/Mean) * 100
•
The % CV must be less than 3.0 to pass.
Sensitivity Test
1.
Calculate the Mean and Standard Deviation for the buffer wells (C10-F12).
2.
Calculate the Mean for the 1000 pM (1.0 nM) SF solution wells (C1-F1).
3.
Calculate the Detection Limit, in pg/mL:
1000 / ((Mean SF - Mean Buffer)/(3 * Standard Deviation Buffer))
Optic Probe
Detection Limit must be less than:
Bottom 5 mm
26 pM (10 pg/mL)
Top 3 mm
53 pM (20 pg/mL)
Linearity Test
1.
Calculate the Mean of the four wells for each concentration in columns 1-5 (rows CF only).
2.
Perform linear regression using these values as inputs:
x
1000
3.
y
mean of the 1000 pM (1.0 nM) wells
500
mean of the 500 pM (0.5 nM) wells
250
mean of the 250 pM (0.25 nM) wells
125
mean of the 125 pM (0.125 nM) wells
62.5
mean of the 62.5 pM (0.0625 nM) wells
Calculate the R-Squared value; it must be greater than or equal to 0.950 to
pass.
FLx800 Operator’s Manual
114 | Chapter 5: Instrument Qualification
Troubleshooting
If any tests fail, please try the suggestions below. If the test(s) continue to fail, print the
results and contact BioTek’s Technical Assistance Center.
•
Are the solutions fresh? The open buffer and stock solutions should be discarded
after seven days.
•
Are the Excitation/Emission filters clean? Are they in the proper locations and in
the proper orientation in the filter wheels?
•
Are you using new/clean plates? We suggest you re-run the test with a new/clean
microplate.
 For bottom optics tests, if the base of a plate is touched, clean the entire base with
alcohol (95% ethanol) and then wipe with a lint-free cloth. Before placing a plate
in the instrument, blow the bottom of the plate with an aerosol.
 The Methylumbelliferone tests are very sensitive to particulates in the wells. If
your tests are failing, you may want to try using sterile microplates. (The plates
recommended under “Required Materials” are not sterile.)
 If the test continues to fail, the optical probe(s) may need to be cleaned. See the
instructions in Chapter 6: Preventive Maintenance. If a test fails because one
or more wells overranged, reduce the Sensitivity value in the protocol by 1-5
counts and re-read the plate.
•
If the Corners Test continues to fail, the hardware may be misaligned. Contact
BioTek TAC.
•
Review the instructions under “Pipette Map” (page 112) to verify that you correctly
prepared the plates.
•
If you manually transferred data from a printout or data file into the spreadsheets,
verify that that each spreadsheet cell contains the correct well value.
BioTek Instruments, Inc.
Corners, Sensitivity, and Linearity Tests |
115
Gen5 Protocol Reading Parameters
These tables contain the reading parameters for the tests detailed in this chapter. Your tests
may require modifications to some of the parameters, such as the Gain/Sensitivity. For
parameters not shown, default values are used.
Gen5 Protocol Name: ‘FLx800 FI_B’
Parameter
Setting
Plate Type:
96 WELL PLATE
Read Wells:
Corners Read step: All wells
Sensitivity Read step: Wells C1-F12
Filters:
EX 485/20 nm, EM 528/20 nm
Optics Position:
Bottom
Gain/Sensitivity:
Corners Read step: 80
Sensitivity/Linearity Read step: 100
Delay After Plate Movement:
350 msec
Measurements Per Data Point:
40
Gen5 Protocol Name: ‘FLx800 FI_T’
Parameter
Setting
Plate Type:
96 WELL PLATE
Read Wells:
Corners Read step: All wells
Sensitivity Read step: Wells C1-F12
Filters:
EX 485/20 nm, EM 528/20 nm
Optics Position:
Top
Gain/Sensitivity:
Corners Read step: 90
Sensitivity/Linearity Read step: 100
Delay After Plate Movement:
350 msec
Measurements Per Data Point:
40
FLx800 Operator’s Manual
116 | Chapter 5: Instrument Qualification
Onboard Assay Parameters
These tables contain the recommended reading parameters if you are operating the FLx800
via the keypad. Your tests may require modifications to some of the parameters, such as
the Gain/Sensitivity (see “Troubleshooting” on page 114). For parameters not shown, use
default values.
Assay Name: ‘FLBOT’
Parameter
Setting (METHOD)
Assay Type:
FLUOR
Plate Type:
96 WELLS
Wavelength:
SINGLE
Number of Filter Sets:
2
Eject Between Sets:
NO
For both Filter Sets unless specified otherwise:
Probe:
BOTTOM
EX/EM:
485/20 nm, 528/20 nm
Samples Per Well:
040
Delay Before Samples:
0350 ms
Delay Between Samples:
001 ms
Gain/Sensitivity:
080 (Filter Set #1), 100 (Filter Set #2)
Assay Name: ‘FLTOP’
Parameter
Setting (METHOD)
Assay Type:
FLUOR
Plate Type:
96 WELLS
Wavelength:
SINGLE
Number of Filter Sets:
2
Eject Between Sets:
NO
For both Filter Sets unless specified otherwise:
Probe:
TOP
EX/EM:
485/20 nm, 528/20 nm
Samples Per Well:
040
Delay Before Samples:
0350 ms
Delay Between Samples:
001 ms
Gain/Sensitivity:
090 (Filter Set #1), 100 (Filter Set #2)
BioTek Instruments, Inc.
Corners, Sensitivity, and Linearity Tests |
117
Using Methylumbelliferone
As an alternative to using Sodium Fluorescein, Methylumbelliferone (“MUB”) can be used
to test the top optics for the fluorescence system.
Required Materials
 BioTek offers a liquid test kit (PN 7160012) containing the microplate and
solutions used in this procedure.
 Microplates should be perfectly clean and free of dust or bottom scratches.
Use new microplates from sealed packages.
 FLx800 IBEP model: To effectively read microplates using the top
probe, a plate carrier adapter is required (BioTek PN 7080522)
to raise the microplate and bring it closer to the probe. Microplates with
heights up to 0.600" can be read using this adapter.
•
A new, clean, 96-well solid black plate, such as Corning® Costar Mfr. #3915.
•
Excitation filter 360/40 nm, Emission filter 460/40 nm
•
Deionized or distilled water
•
Carbonate-Bicarbonate buffer (“CBB”) capsules (BioTek PN 98158)
•
10 mg vial of Methylumbelliferone (MUB) (BioTek PN 98156)
•
100% methanol (BioTek PN 98161)
•
Aluminum foil
•
Various beakers, graduated cylinders, and pipettes
•
(Optional, but recommended) 0.45 micron filter
•
Assays and reading parameters
FLx800 Operator’s Manual
118 | Chapter 5: Instrument Qualification
Test Solutions

We recommend that you filter solutions to remove particulates
that could cause erroneous readings. Do not allow dust to settle on
the surface of the solution; use microplate covers or seals when not
reading the plate.
Wrap the vial containing the MUB stock solution in foil to prevent
exposure to light.
Discard any open, unused solutions after seven days.
1.
2.
3.
Prepare the buffer (CBB) solution:
•
(Optional, but recommended) Using a 0.45-micron filter, filter 200 mL of
deionized or distilled water.
•
Open and dissolve the contents of 2 CBB capsules (do not dissolve the outer
gelatin capsule) into 200 mL of the water.
•
Stir the solution (preferably using a stir table) until the CBB is completely
dissolved.
Prepare the MUB stock solution:
•
Add 1 mL of 100% methanol to the 10 mg vial of MUB.
•
Make sure all of the dye has completely dissolved and is well mixed. This
yields a 10 mg/mL stock solution.
•
Wrap the solution in aluminum foil to prevent exposure to light.
Prepare the dilutions. Label each with “MUB” and the concentration.
Mix this MUB solution:
with:
to make:
0.5 mL of 10 mg/mL stock solution
4.5 mL of 100%
methanol
1 mg/mL
0.88 mL of 1 mg/mL solution
4.12 mL of CBB
176 μg/mL
0.1 mL of 176 μg/mL solution
9.9 mL of CBB
1.76 μg/mL
0.5 mL of 1.76 μg/mL solution
4.5 mL of CBB
176 ng/mL
1 mL of 176 ng/mL solution
9 mL of CBB
17.6 ng/mL
(100 nM)
BioTek Instruments, Inc.
Corners, Sensitivity, and Linearity Tests |
119
Procedure – Gen5
1.
If you have not already done so, prepare the test solutions. See page 118.
2.
If you have not already done so, create the Gen5 protocol ‘FLx800 FI_MUB’
described on page 122.
3.
Adjust the probe height for the plate being used; the probe should be lowered as
close to the plate as possible without interfering with its movement. See Top
Optical Probe Adjustment in Chapter 2.
4.
Pipette the test solutions into a new 96-well solid black microplate (see the map on
page 120).
5.
Create a Gen5 experiment based on ‘FLx800 FI_MUB’ and read the plate.
6.
When complete, save the experiment using a unique name.
7.
Analyze the data using the calculations on page 121.
Procedure – Keypad
1.
Make sure the printer is attached to the reader and turned on. Configure the reader
to print results in the Matrix Report format.
2.
If you have not already done so, create the ‘FLMUB’ assay described on page 122.
3.
If you have not already done so, prepare the test solutions; see page 118.
4.
Adjust the probe height for the plate being used; the probe should be lowered as
close to the plate as possible without interfering with its movement. See Top
Optical Probe Adjustment in Chapter 2.
5.
Pipette the test solutions into a new 96-well solid black microplate (see the map on
page 120).
6.
Read the plate using the ‘FLMUB’ assay. When prompted, enter 96 for the number
of samples.
7.
When finished, print the report.
8.
Analyze the data using the calculations on page 121.
FLx800 Operator’s Manual
120 | Chapter 5: Instrument Qualification
Pipette Map for Methylumbelliferone Tests
 Seal the plate with foil or store it in a black polyethylene bag until use.
Use a multi-channel pipette with just 4 tips installed, for pipetting into rows C, D, E,
and F. Perform these instructions carefully, and refer to the grid below.
1
•
Pipette 150 µL of CBB buffer into columns 2-5 and 10-12.
•
Pipette 150 µL of the 17.6 ng/mL (100 nM) MUB solution into column 1.
Discard the tips.
•
Pipette 150 µL of the 17.6 ng/mL solution into column 2.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 2 and dispense it into column 3.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 3 and dispense it into column 4.
Mix the wells using the pipette. Do not discard the tips.
•
Aspirate 150 µL from column 4 and dispense into column 5.
Mix the wells using the pipette.
•
Aspirate 150 µL from column 5 and discard.
2
3
4
5
6
7
8
9
10
11
12
A
A
B
B
C
100
nM
50
nM
25
nM
12.5
nM
6.25
nM
BUF
BUF
BUF
C
D
100
nM
50
nM
25
nM
12.5
nM
6.25
nM
BUF
BUF
BUF
D
E
100
nM
50
nM
25
nM
12.5
nM
6.25
nM
BUF
BUF
BUF
E
F
100
nM
50
nM
25
nM
12.5
nM
6.25
nM
BUF
BUF
BUF
F
G
G
H
H
BioTek Instruments, Inc.
Corners, Sensitivity, and Linearity Tests |
121
Results Analysis
Sensitivity Test
1. Calculate the Mean and Standard Deviation for the buffer wells (C10-F12).
2.
Calculate the Mean for the 17.6 ng/mL (100 nM) MUB solution wells
(C1-F1).
3.
Calculate the Detection Limit, in ng/mL:
17.6 / ((Mean MUB - Mean Buffer)/(3 * Standard Deviation Buffer))
Optic Probe
Detection Limit must
be less than:
Top 3 mm
0.16 ng/mL
Linearity Test
1. Calculate the Mean of the four wells for each concentration in columns 1-5.
2.
Perform linear regression using these values as inputs:
x
100
3.
y
mean of the 100 nM wells
50
mean of the 50 nM wells
25
mean of the 25 nM wells
12.5
mean of the 12.5 nM wells
6.25
mean of the 6.25 nM wells
Calculate the R-Squared value; it must be greater than or equal to 0.950 to
pass.
FLx800 Operator’s Manual
122 | Chapter 5: Instrument Qualification
Gen5 Protocol Reading Parameters
This table contains the reading parameters for the test described in this chapter. Your
tests may require modifications to some of the parameters, such as the Gain/Sensitivity
(see “Troubleshooting” on page 114). For parameters not shown, default values are
used.
Protocol Name: ‘FLx800 FI_MUB.prt’
Parameter
Setting
Plate Type:
96 WELL PLATE
Read Wells:
Wells C1 to F12
Detection Method:
Fluorescence
Read Type:
Endpoint
Filters:
EX 360/40 nm, EM 460/40 nm
Optics Position:
Top
Gain/Sensitivity:
90
Delay After Plate Movement:
350 msec
Measurements Per Data Point:
40
Onboard Assay Parameters
This table contains the recommended reading parameters if you are operating the
FLx800 via the keypad. Your tests may require modifications to some of the parameters,
such as the Sensitivity (see “Troubleshooting” on page 114). For parameters not shown,
use default values.
Assay Name: ‘FLMUB’
Parameter
Setting
Assay Type:
FLUOR
Plate Type:
96 WELLS
Wavelength:
SINGLE
Number of Filter Sets:
1
Probe:
TOP
EX/EM:
360/40 nm, 460/40 nm
Samples Per Well:
040
Delay Before Samples:
0350 ms
Delay Between Samples:
001 ms
Gain/Sensitivity:
090
BioTek Instruments, Inc.
Dispense Accuracy & Precision Tests |
123
Dispense Accuracy & Precision Tests
This section applies to models with the dispense module only.
BioTek Instruments, Inc. has developed a set of tests to ensure that your dispense module
performs to specification initially and over time. We recommend that you perform these tests
before first use (e.g., during the Initial OQ), and then every three months.
•
The Accuracy Test measures the mean volume per well for multiple dispenses. The
actual weight of the dispensed fluid is compared to the expected weight and must be
within a certain percentage to pass. Pass/Fail criteria depends on the per-well volume
dispensed. The test uses a green dye test solution and one 96-well microplate to test
the three different volumes. The precision balance is tared with the empty plate, and
then the 80 µL dispense is performed for columns 1-4. The fluid is weighed and the
balance is tared again (with the plate on the balance). This process is repeated for the
20 µL and 5 µL dispenses. It is assumed that the solutions used are at room
temperature, and one gram is equal to one milliliter.
•
The Precision Test measures the variation among volumes dispensed to multiple
wells. For each volume dispensed (80 µL, 20 µL, and 5 µL) to four columns, the %CV
(coefficient of variation) of 32 absorbance readings is calculated. Pass/Fail criteria
depends on the per-well volume dispensed. The plate is read in an absorbance reader
at 405/750 nm for columns 1-4 and at 630/750 nm for columns 5-12. The two tests are
performed simultaneously and use the same plate.
Required Materials
•
Absorbance reader with capability of measuring 96-well microplates at 405, 630, and
750 nm. The reader must have an accuracy specification of ± 1.0% ± 0.010 OD or better
and a repeatability specification of ± 1.0% ± 0.005 OD or better.
•
Microplate shaker (if the absorbance reader does not support shaking)
•
Precision balance with capacity of 100 g minimum and readability of 0.001 g
•
50-200 µL hand pipette and disposable tips
•
Deionized water
•
Supply bottle
•
250 mL beaker
•
New 96-well, clear, flat-bottom microplate
•
BioTek’s Green Test Dye Solution (PN 7773003) undiluted, or one of the alternate test
solutions supplied on the next page
•
100 mL graduated cylinder and 10 mL pipettes (if not using BioTek’s Green Test Dye
Solution)
FLx800 Operator’s Manual
124 | Chapter 5: Instrument Qualification
•
Gen5 software installed on the host PC
•
Calculation worksheet at the end of this chapter
Alternate Test Solutions
 80 µL of test solution with 150 µL of deionized water should read
between 1.300 and 1.700 OD at 405/750 nm. It is assumed that the
solutions used are at room temperature.
If you do not have BioTek’s Green Test Dye Solution (PN 7773003), prepare a green test
dye solution using one of the following methods:
Using BioTek’s Blue and Yellow Concentrate Dye Solutions:
Ingredient
Quantity
Concentrate Blue Dye Solution (PN 7773001, 125 mL)
4.0 mL
QC (Yellow) Solution (PN 7120782, 125 mL)
5.0 mL
Deionized water
90.0 mL
Using FD&C Blue and Yellow Dye Powder:
Ingredient
Quantity per Liter
FD&C Blue No. 1
0.200 grams
FD&C Yellow No. 5
0.092 grams
Tween 20®
Sodium Azide N3Na
Deionized water
1.0 mL
0.100 gram
Make to 1 liter
BioTek Instruments, Inc.
Dispense Accuracy & Precision Tests |
125
Test Setup
To perform the Dispense Accuracy and Precision Test using Gen5, you will need to create
a protocol containing the necessary Dispense steps. If you will be using one of BioTek’s
absorbance readers (see Required Materials on page 123), you can optionally create a
second protocol containing the necessary Read steps. You will create the protocol(s) once
and then reference them in new experiments each time you run the test. See page 128 for
instructions for creating the protocols.
If you are not using a BioTek absorbance reader, or if you prefer to operate your BioTek
reader using its keypad, prepare the reader to perform two reads with the following
characteristics:
For the 80 µl Read
For the 20 and 5 µl Read
Primary Wavelength
405 nm
630 nm
Reference Wavelength
750 nm
750 nm
Plate Columns
1-4
5-12
•
For your convenience, we’ve included a worksheet at the end of this chapter for
recording the dispense weights, Delta OD values, calculations, and pass/fail.
Procedure
 If you are using one of BioTek’s keypad-based absorbance readers, such as the
ELx800 or ELx808, make sure the reader is not running in Rapid mode. To check
the setting, select UTIL > READ and cycle through the options until READ IN
RAPID MODE? appears. Set it to NO.
1.
Turn on the FLx800 and the absorbance reader.
2.
Start Gen5 and, if necessary, configure the software to communicate with the
FLx800 (System > Reader Configuration). If you will be using a BioTek
absorbance reader and your Gen5 version supports the use of two readers at the
same time, configure Gen5 for the absorbance reader as well.
3.
Prime the system with deionized or distilled water:
•
Ensure that the tip priming trough (BioTek PN 7082118) is installed in the
microplate carrier.
•
Place a clean priming plate (BioTek PN 7132158 or 7092135) on the carrier.
•
Fill a supply bottle with deionized or distilled water and insert the inlet tube.
•
In Gen5, select System > Reader Control. If prompted, select the reader
under test.
•
Click the Dispenser tab. If the ‘Initialized’ status is “No” click Initialize.
FLx800 Operator’s Manual
126 | Chapter 5: Instrument Qualification
•
Enter a prime Volume of 4000 µL and click Prime.
•
When finished remove the plate, empty it, and put it back on the carrier.
4.
Remove the inlet tube from the supply bottle. Prime the dispenser again with the
5.
Fill a supply bottle or beaker with at least 20 mL of the green dye solution. Prime
the dispenser with 2000 mL of the solution. When finished, remove the priming
plate from the carrier.
6.
Create an experiment for the FLx800 based on the Dispense Protocol described
at the end of this chapter.
7.
Place a new 96-well microplate on the balance and tare the balance.
8.
Place the plate on the FLx800’s microplate carrier.
9.
Initiate the plate read. Gen5 will prompt you to empty the tip priming trough (do
so if necessary).
Volume set to 2000 µL. This prevents the water from diluting the dye solution.
10. When ready, begin the experiment. Follow the prompts displayed on the screen;
the sequence is as follows:
•
Dispense 80 µL/well to columns 1-4.
•
Remove the plate and weigh it. Record the weight and tare the balance.
•
Place the plate on the carrier, dispense 20 µL/well to columns 5-8.
•
Remove the plate and weigh it. Record the weight and tare the balance.
•
Place the plate on the carrier, dispense 5 µL/well to columns 9-12.
•
Remove the plate and weigh it. Record the weight. Set the plate aside.
•
Finish by performing a brief read step (Gen5 requires a Read step in a Dispense
protocol; the measurement value is not used).
11. Manually pipette 150 µL/well of deionized or distilled water into all 12
columns of the plate, on top of the green test dye solution.
12. If the absorbance reader does not support shaking, shake the plate at variable
speed for 15 seconds.
13. Place the plate on the absorbance reader’s microplate carrier.
14. If you are using Gen5 to control the absorbance reader:
•
Create an experiment for the absorbance reader based on the Read Protocol
described at the end of this chapter.
•
Initiate the plate read. Follow the prompts on the screen.
•
When processing is complete, save the file with an identifying name.
If you are not using Gen5 to control the absorbance reader:
•
Read columns 1-4 at 405/750 nm.
BioTek Instruments, Inc.
Dispense Accuracy & Precision Tests |
15.
•
Read columns 5-12 at 630/750 nm.
•
When processing is complete, save or print the results.
127
See page 127 for instructions for analyzing the results.
16. When all tests are complete, prime the dispenser with at least 5000 µL of deionized
or distilled water, to flush out the green dye solution.
Results Analysis
1.
2.
Choose one of the following methods for recording the data and performing the
analysis:
•
Make a copy of the worksheet supplied at the end of this section and enter the data
manually.
•
Export the data to your own analysis or spreadsheet program.
If you are performing data reduction manually, calculate the following for each volume
dispensed (80, 20, 5 µL):
•
Calculate the Standard Deviation of the 32 wells.
•
Calculate the Mean of the 32 wells.
•
Calculate the %CV: abs(Standard Deviation / Mean) * 100
•
Calculate the Accuracy % Error:
abs((Actual Weight - Expected Weight) / Expected Weight) * 100
 Expected Weights for 32 wells: 80 μL (2.560 g), 20 μL (0.640 g),
5 μL (0.160 g). It is assumed that one gram is equal to one milliliter.
Dispense
Volume
To pass,
% CV must be:
To pass, Accuracy
% Error must be:
80 µL
≤ 2.0%
≤ 2.0%
20 μL
≤ 7.0%
≤ 5.0%
5 μL
≤ 10.0%
≤ 20.0%
If any tests fail, prime the fluid lines and re-run the test(s). If the test(s) fail again, the
injector head may require cleaning (see Section 5, Preventive Maintenance). If tests
continue to fail, contact BioTek’s Technical Assistance Center.
FLx800 Operator’s Manual
128 | Chapter 5: Instrument Qualification
Gen5 Test Protocols
This section contains instructions for creating a Gen5 protocol to dispense three different
volumes of fluid into varying columns in the microplate. If you will be using one of BioTek’s
absorbance readers, you can optionally create a second protocol containing the necessary read
steps.
The Dispense protocol follows the sequence below. After each Dispense step, the plate is
ejected to allow the operator to weigh it and then tare the balance.
•
Dispense 80 µl dye to columns 1-4
•
Dispense 20 µl dye to columns 5-8
•
Dispense 5 µl dye to columns 9-12
The optional Read protocol follows this sequence:
•
Shake the plate for 15 seconds
•
Read columns 1-4 at 405/750 nm, calculate the Delta OD
•
Read columns 5-12 at 630/750 nm, calculate the Delta OD
To create the Dispense protocol (required):
1.
From Gen5’s main screen, select System > Reader Configuration, and
add/configure the FLx800 (if it is not already there).
2.
Create a new protocol.
3.
Select Protocol > Procedure. If prompted to select a reader, select the FLx800. The
Procedure dialog will open.
4.
Click the Dispense button to open the Dispense Step dialog. Define the following
characteristics:
•
Dispense to wells A1-H4
•
Set Tip Prime to None
•
Dispense 80 µl/well at a rate of 275 µl/second
5.
Click OK to add the step to the Procedure.
6.
Click the Plate Out/In button to open the Plate Out/In Step dialog.
7.
Select Move plate out, display dialog, move plate in, and enter the following text
in the Comment box:
“Weigh the plate (80 ul test). RECORD the weight, TARE the balance. Place the plate
back on the carrier. Click OK to continue.”
8.
Click OK to add the step to the Procedure.
9.
Add another Dispense step with the following characteristics:
•
Dispense to wells A5-H8
BioTek Instruments, Inc.
Dispense Accuracy & Precision Tests |
•
Set Tip Prime to before this dispense step, Volume 20 µl
•
Dispense 20 µl/well at a rate of 250 µl/second
129
10. Add another Plate Out/In step with the following Comment:
“Weigh the plate (20 ul test). RECORD the weight, TARE the balance. Place the plate
back on the carrier. Click OK to continue.”
11.
Add another Dispense step with the following characteristics:
•
Dispense to wells A9-H12
•
Set Tip Prime to before this dispense step, Volume 5 µl
•
Dispense 5 µl/well at a rate of 225 µl/second
12. Add another Plate Out/In step with the following Comment:
“Weigh the plate (5 ul test). RECORD the weight. Set the plate aside and click OK.”
13.
The last step is a brief Read step. This step is necessary because Gen5 requires a Read
step within any Dispense procedure. When this test is run, the measurement value is not
used.
Click the Read button to open the Read Step dialog. We recommend defining the
following characteristics, although you can change any of the settings:
•
Read well A1 only
•
Detection Method: Luminescence
•
Emission: Select any available plug, filter, or open location, preferably one
that will remain in use in this instrument.
•
Optics Position: Top
•
Sensitivity: 0
14. Click OK to add the step to the procedure. The procedure should resemble the
following:
15.
Make any changes if necessary. Click OK to save the procedure.
16. Save the protocol with an identifying name, such as FLx800 Accuracy and Precision
- Dispense.prt.
FLx800 Operator’s Manual
130 | Chapter 5: Instrument Qualification
To create the Read protocol (optional):
1.
Select System > Reader Configuration, and add/configure the BioTek absorbance
reader (if it is not already there).
2.
Create a new protocol.
3.
Select Protocol > Procedure. If prompted to select a reader, select the absorbance
reader. The Procedure dialog will open.
4.
Click the Shake button to open the Shake Step dialog. Define the following
characteristics:
•
Intensity: Medium
•
Duration: 0:15 (MM:SS)
5.
Click OK to add the step to the procedure.
6.
Click the Read button to open the Read step dialog. Define the following characteristics:
•
Step label: ‘80 ul Read’
•
Read wells A1 to H4 only
•
Detection Method: Absorbance
•
Read Type: Endpoint
•
Read Speed: Normal
•
Wavelengths: 405, 750 nm
7.
Click OK to add the step to the procedure.
8.
Click the Read button to open the Read step dialog. Define the following characteristics:
9.
•
Step label: ‘20 and 5 ul Read’
•
Read wells A5 to H12 only
•
Detection Method: Absorbance
•
Read Type: Endpoint
•
Read Speed: Normal
•
Wavelengths: 630, 750 nm
Click OK to add the step to the procedure. The procedure should resemble the
following:
BioTek Instruments, Inc.
Dispense Accuracy & Precision Tests |
10.
Make any changes if necessary. Click OK to save the procedure.
11.
Select Protocol > Data Reduction and select Custom (for Gen5 1.x users, select
Transformation).
131
12. Within this dialog, click the Select Multiple Data Sets button and then click the DS2
radio button (see below).
•
Set the Data In for DS1 to the 80 µl Read step at 405 nm.
•
Set the Data In for DS2 to the 80 µl Read step at 750 nm.
13. Click OK to return to the dialog.
14.
In the New Data Set Name field, type an identifying name such as ‘Delta OD 80 ul’
(see sample screen below).
15. Uncheck Use single formula for all wells.
16.
In the Formula field, type DS1-DS2 and then highlight wells A1 to H4 to assign the
formula.
FLx800 Operator’s Manual
132 | Chapter 5: Instrument Qualification
17.
Click OK to add the transformation to the Data Reduction list.
18.
Create another Transformation with these characteristics:
•
DS1 set to the 20 and 5 µl Read step at 630 nm
•
DS2 set to the 20 and 5 µl Read step at 750 nm
•
New Data Set Name resembling ‘Delta OD 20 and 5 ul’
•
Formula DS1-DS2 applied to wells A5 to H12
When you’re finished, the Data Reduction Steps list will show two Delta OD
transformations:
19.
Click OK to close the Data Reduction dialog.
20. Save the protocol with an identifying name, such as FLx800 Accuracy and Precision Read.prt.
BioTek Instruments, Inc.
FLx800 Dispense Accuracy & Precision Test
80 µl Dispense
Delta ODs @405/750 nm
1
2
3
20 µl Dispense
Delta ODs @630/750 nm
4
5
6
7
5 µl Dispense
Delta ODs @630/750 nm
8
9
10
11
12
A
A
B
B
C
C
D
D
E
E
F
F
G
G
H
H
80 µl weight:
g
20 µl weight:
g
5 µl weight:
g
Expected weight:
2.5600 g
Expected weight:
0.6400 g
Expected weight:
0.1600 g
%
Accuracy % Error:
Must be <= 2.0%
P
F
%
Accuracy % Error:
Must be <= 5.0%
P
F
Must be <= 20.0%
Standard Deviation:
Standard Deviation:
Standard Deviation:
Mean:
Mean:
Mean:
%
%CV:
Must be <= 2.0%
P
F
%
%CV:
Must be <= 7.0%
Reader Model:
P
F
P
F
%
%CV:
Must be <= 10.0%
Reader S/N:
Tested By:
Reviewed/
Approved By:
Reading Date:
Signature:
Signature:
Comments:
%
Accuracy % Error:
P
F
Chapter 6
Preventive Maintenance
This chapter provides step-by-step instructions for maintaining the
FLx800 and external dispenser module (if used) in top condition, to
ensure that they continue to perform to specification.
Recommended Maintenance Schedule ............................... 134
Overview................................................................... 134
Dispenser Module ....................................................... 134
Schedule ................................................................... 134
Warnings & Precautions ................................................... 136
Cleaning Exposed Surfaces .............................................. 137
Inspecting/Cleaning Excitation and Emission Filters ............. 138
Flushing/Purging the Fluid Path ........................................ 139
Running a Dispense Protocol (Optional) ............................. 140
Emptying/Cleaning the Tip Prime Trough ........................... 141
Cleaning the Supply Bottles.............................................. 141
Cleaning the Priming Plate ............................................... 141
Cleaning the Internal Components .................................... 142
Required Materials ...................................................... 143
Removing the Reader’s Cover ...................................... 144
Cleaning the Optical Probe(s)....................................... 145
Removing/Cleaning the Internal Dispense Tube and Injector
Head ........................................................................ 148
Cleaning the Internal Instrument Surface ...................... 150
Reassembling the Components .................................... 151
Performance Check..................................................... 151
134 | Chapter 6: Preventive Maintenance
Recommended Maintenance Schedule
Overview
A general Preventive Maintenance (PM) regimen for all FLx800 models includes
periodically cleaning all exposed surfaces, inspecting/cleaning the Excitation and
Emission filters, and cleaning the optical probe(s). For models with the external dispenser
module, additional tasks include flushing/purging the fluid path and cleaning the tip
prime trough, priming plate, supply bottle, internal dispense tubing, and injector head.
Dispenser Module
A daily cleaning regimen is the best way to ensure accurate performance and a long life for
your instrument and dispenser module. To keep the dispenser module and injector in top
condition, flush and purge the system with deionized (DI) water every day or upon
completion of an assay run, whichever is more frequent.
Some reagents may crystallize or harden after use, clogging the fluid passageways.
Flushing the tubing at the end of each day, letting the DI water soak the tubing overnight,
and then purging the lines at the beginning of each day ensures optimal performance of
the dispense system. Perform a visual inspection of the dispensing accuracy before
conducting an assay that requires dispense to verify instrument performance.
It is important to keep the dispensing lines scrupulously clean at all times. Take special
care when using molecules active at very low concentrations (e.g., enzymes, inhibitors).
Remove any residual reagent in the dispensing lines using a suitable cleaning solution
(review the reagent’s package insert for specific recommendations).
 BioTek also recommends flushing the module with DI water before
conducting the decontamination procedure described in Appendix A.
Schedule
The following charts recommend Preventive Maintenance tasks and the frequency with
which each task should be performed.

It is important to note that the risk and performance
factors associated with your assays may require that some
or all of the procedures be performed more frequently
than presented in the schedule.
BioTek Instruments, Inc.
Recommended Maintenance Schedule |
Tasks for FLx800 Models
without the Dispenser Module
Frequency
Quarterly
Annually
As Needed

Clean Exposed Surfaces
Inspect/Clean Excitation and Emission Filters

Clean Optical Probes

Clean Internal Instrument Surface

Decontamination

Before shipment or storage
Tasks for FLx800 Models
with the Dispenser Module
Frequency
Daily
Quarterly
As Needed

Clean Exposed Surfaces

Inspect/Clean Excitation and Emission Filters
Flush/Purge the Fluid Path


(Optional) Run Dispense Protocol
Empty/Clean Tip Prime Trough

Clean Supply Bottles

Clean Priming Plate

Clean Optical Probes

Clean Tubing and Injector Head

Clean Internal Instrument Surface

Decontamination

Before shipment or storage
Accumulated algae, fungi, or mold may require
decontamination. See Appendix A for complete
decontamination instructions for all models.
FLx800 Operator’s Manual

135
136 | Chapter 6: Preventive Maintenance
Warnings & Precautions
Please read the following before performing any maintenance procedures:
Warning! Internal Voltage. Turn off and unplug
the instrument for all maintenance and repair
operations.
Warning! Wear protective gloves when handling
contaminated instruments. Gloved hands should be
considered contaminated at all times; keep gloved
hands away from eyes, mouth, nose, and ears.
Warning! Mucous membranes are considered prime
entry routes for infectious agents. Wear eye protection
and a surgical mask when there is a possibility of
aerosol contamination. Intact skin is generally
considered an effective barrier against infectious
organisms; however, small abrasions and cuts may not
always be visible. Wear protective gloves when
handling contaminated instruments.

Important! Do not immerse the instrument, spray it

Important! Do not apply lubricants to the microplate
with liquid, or use a “wet” cloth on it. Do not allow
water or other cleaning solution to run into the interior
of the instrument. If this happens, contact BioTek’s
Technical Assistance Center.
carrier or carrier track. Lubrication on the carrier
mechanism or components in the carrier compartment
will attract dust and other particles, which may
obstruct the carrier path and cause the instrument to
produce an error.
Caution! The buildup of deposits left by the
evaporation of spilled fluids within the read chamber
can impact fluorescence measurements. Be sure to
keep System Test records before and after maintenance
so that changes can be noted.
BioTek Instruments, Inc.
Cleaning Exposed Surfaces |
137
Caution! Models with the external dispenser module.
Before removing the cover to expose internal parts,
purge the dispenser module, turn off the instrument,
and disconnect the fluid line, power cable, and PC
cable.
Cleaning Exposed Surfaces
Warning! Turn off and unplug the instrument for all
cleaning operations.
Important! Do not immerse the instrument, spray it
with liquid, or use a “wet” cloth. Do not allow the
cleaning solution to run into the interior of the
instrument. If this happens, contact BioTek’s Service
Department.
To clean the FLx800’s exposed surfaces:
1.
Turn off and unplug the reader.
2.
If fluid has spilled inside the instrument:
•
See page 144 for instructions for removing the cover.
•
See page 150 for instructions for cleaning the internal instrument surface.
3.
Moisten a clean cotton cloth with water, or with water and mild detergent. Do not soak
4.
Wipe the plate carrier and all exposed surfaces of the instrument. If the dispenser
module is attached, wipe its exposed surfaces as well.
5.
If detergent was used, wipe all surfaces with a cloth moistened with water.
6.
Use a clean, dry cloth to dry all wet surfaces.
7.
Reassemble the instrument as necessary.
the cloth.

FLx800 Operator’s Manual
Models with the external dispenser module:
If the Tip Prime Trough overflows, wipe the carrier
and the surface beneath the carrier with a dry cotton
cloth. If overflow is significant, you may have to
remove the reader’s cover and lift the carrier to better
clean its bottom surface. See Cleaning the Internal
Components starting on page 150 for instructions.
138 | Chapter 6: Preventive Maintenance
Inspecting/Cleaning Excitation and Emission Filters
Laboratory air is used to cool the lamp, and the filters can become dusty as a result. The filters
should be inspected and cleaned at least every three months.
Important: Do not touch the filters with your bare fingers!
To inspect and clean the Excitation and Emission filters:
1.
Obtain lens cleaning tissue and isopropyl, ethyl, or methyl alcohol.
2.
Turn off and unplug the reader.
3.
Pull down the hinged door on the front of the reader. Observe the two thumbscrews
within the compartment. The left thumbscrew holds the excitation (EX) filter wheel in
place; the right secures the emission (EM) filter wheel. Remove each thumbscrew and
slide the filter wheel’s supporting metal bracket straight out of the compartment.
 Chapter 2, Instrument Description contains some illustrations that
may be useful for identifying the filter wheels and their unique
characteristics. This chapter also contains instructions for replacing filters
if necessary.
4.
Inspect the glass filters for speckled surfaces or a halo effect. This may indicate
deterioration due to moisture exposure over a long period of time. If you have any
concerns about the quality of the filters, contact your BioTek representative.
5.
Clean the filters using lens-cleaning tissue moistened with a small amount of isopropyl,
ethyl, or methyl alcohol. Ensure that the filters remain in their current locations.
6.
Replace the filter wheel brackets in their respective positions and replace the
thumbscrews. Close the hinged door.
BioTek Instruments, Inc.
Flushing/Purging the Fluid Path |
139
Flushing/Purging the Fluid Path
Applies only to models with the external dispenser module.
At the end of each day that the dispenser module is in use, flush the fluid path using Gen5’s
priming utility. Leave the fluid to soak overnight or over a weekend, and then purge the fluid
before using the reader again.
Note: This flushing and purging routine is also recommended before decontamination, to
remove any residue from the fluid lines before applying isopropyl alcohol. See Appendix A
for complete decontamination instructions.
To flush the fluid path:
1.
Fill the reagent bottle with distilled or deionized water. Insert the supply tube into the
bottle.
2.
Place the priming plate on the microplate carrier.
3.
Access Gen5’s main screen and select System > Reader Control > FLx800.
4.
Click the Dispenser tab.
5.
Initialize the dispenser if necessary.
6.
Set the Volume to 5000 μl. Keep the default Dispense Rate.
7.
Click Prime
8.
When the prime is complete, carefully remove the priming plate from the carrier and
empty it.
Leave the water in the system overnight, or until the reader will be used again. Purge the fluid
from the system (see below) and then prime with the injection reagent before running an
assay.
To purge the fluid from the system:
1.
Exchange the reagent bottle with a waste vessel.
2.
Repeat steps 3-5 above
3.
Set the Volume to 2000 μl.
4.
Click Purge
5.
When the purge is complete, empty the waste vessel.
After flushing/purging the system, you may wish to run a quick Dispense protocol to visually
verify the dispense accuracy. See the next page for instructions.
FLx800 Operator’s Manual
140 | Chapter 6: Preventive Maintenance
Running a Dispense Protocol (Optional)
Applies only to models with the external dispenser module.
After flushing/purging the system (previous page) and before running an assay that requires
injection, take a moment to visually inspect the dispensing accuracy.

Use a DI H2O-Tween solution to check for dispense
accuracy following maintenance (e.g., add 1 ml
Tween® 20 to 1000 ml of deionized water).
To create the Dispense protocol in Gen5:
1.
Create a new Protocol and then select Protocol > Procedure.
2.
Add a Dispense step with the following parameters:
Tip Priming:
Before this dispense step
Tip Prime Volume: 10 µl
Dispense Volume: 100 µl (or an amount to match your assay protocol)
Rate:
3.
Adjust the rate to support the dispensing volume
Add a brief Read step. This step is necessary because Gen5 requires a Read step within
any Dispense procedure. When this test is run, the measurement value is not used. We
recommend defining the following characteristics, although you can change any of the
settings:
Read well A1 only
Detection Method: Luminescence
Emission:
Select any available plug, filter, or open location, preferably
one that will remain in use in this instrument.
Optics Position:
Top
Gain/Sensitivity:
0
4.
Click OK to close the Procedure.
5.
Save the protocol and give it an identifying name, such as “Dispense Observation.”
BioTek Instruments, Inc.
Emptying/Cleaning the Tip Prime Trough |
141
Emptying/Cleaning the Tip Prime Trough
Applies only to models with the external dispenser module.
The tip prime trough is a small, removable priming cup located in the right rear of the
microplate carrier, used for performing the Tip Prime. The trough holds up to 2 ml of liquid
and must be periodically emptied and cleaned by the user.
To empty/clean the tip prime trough:
1.
Carefully remove the tip prime trough from its pocket in the right rear of the microplate
carrier.
2.
Wash the trough in hot, soapy water. Use a small brush to clean in the corners.
3.
Rinse the trough thoroughly and allow it to dry completely.
4.
Replace the trough in the microplate carrier.
 Note: If the tip prime trough overflows, wipe the carrier and the surface
below the carrier with a dry cotton cloth. If overflow is significant, you
may have to remove the cover of the instrument and lift the carrier to
wipe its bottom surface. See Cleaning the Internal Components
starting on page 150 for complete instructions.
Cleaning the Supply Bottles
Applies only to models with the external dispenser module.
Clean the supply bottles regularly to prevent bacteria growth. Wash them in hot soapy water,
using a small brush to clean inside if necessary. Rinse thoroughly and allow them to dry
completely.
Cleaning the Priming Plate
Applies only to models with the external dispenser module.
Clean the priming plate regularly to prevent bacteria growth and residue buildup. Wash the
plate in hot soapy water, using a small brush to clean in the corners if necessary. Rinse
thoroughly and allow it to dry completely.
FLx800 Operator’s Manual
142 | Chapter 6: Preventive Maintenance
Cleaning the Internal Components
The FLx800’s internal components that require routine cleaning include:
•
Optical probe(s)
•
Surface beneath the microplate carrier
•
Internal dispense tube and injector head (models with the external dispense module
only)
See the recommended schedule on page 134. For models without the dispenser module,
BioTek recommends cleaning the probes annually. For models with the dispenser module, the
recommended frequency is quarterly. In addition, if fluid has spilled inside the instrument
and/or if an unusually high background signal has been flagged by the assay controls
(typically blanks or negative controls), the optical probes and the surface beneath the
microplate carrier should be cleaned.
The procedures in this section should be performed in succession. Start with Removing the
Reader’s Cover and execute the procedures that meet your needs, in the order in which they
are presented. Finish with Performance Check.
We recommend running a System Test before and after performing these cleaning procedures.
This will verify that all systems are functioning properly and allow you to compare results
before and after maintenance.
Caution! The buildup of deposits left by the
evaporation of spilled fluids within the read chamber
can impact fluorescence measurements. Be sure to
keep System Test records before and after maintenance
so that changes can be noted.
BioTek Instruments, Inc.
Cleaning the Internal Components |
143
Required Materials
Warning! Always wear protective gloves and safety
glasses when performing these preventive
maintenance procedures.
For all tasks:
•
Protective gloves
•
Safety glasses
•
Screwdriver
For cleaning the optical probes:
•
Clean cotton swabs
•
Isopropyl alcohol
•
Lens-cleaning tissue
For cleaning the surface under the plate carrier:
•
Isopropyl alcohol
•
Deionized or distilled water
•
Clean, lint-free cotton cloths
•
Compressed air
For cleaning the internal dispense tube and injector head (models with the dispenser module):
•
Mild detergent
•
Deionized or distilled water
•
Stylus (stored in a plastic cylinder affixed to the rear of the dispense module
or reader) (PN 2872304)
FLx800 Operator’s Manual
144 | Chapter 6: Preventive Maintenance
Removing the Reader’s Cover
Caution! Models with the dispenser module: Before
removing the reader’s cover to expose its internal
components, purge the dispenser module, turn off
the instrument, and disconnect the fluid line, power
cable, and PC cable.
1.
If the dispenser module is connected, purge the system of fluid. See page 139 for
instructions. When finished, disconnect the communication cable and the fluid line from
the reader.
2.
Turn off the reader and disconnect the power cable and PC cable (if used).
3.
When the cover is removed, it will be placed upside down and to the right side of the
reader (see the photo on the next page). Clear sufficient space to the right of the reader
to accommodate the cover.
4.
Use a screwdriver to remove the four screws that hold the cover in place, as shown
below. (Contact BioTek TAC if your reader looks different than the one shown here.)
Remove
screws (2)
Remove
screws (2)
Figure 27: Identifying the screws for removing the FLx800’s cover
5.
Models without the dispenser module: Carefully remove the cover by lifting it up off the
reader. The cover will remain connected to the reader via the keypad’s cable. Turn the
cover upside down and rest it to the right of the reader as shown on the next page.
Models with the dispenser module: Carefully remove the cover by lifting the front
edge and sliding the cover toward the rear until it clears the fluid port. The cover will
remain connected to the reader via the keypad’s cable. Turn the cover upside down
and rest it to the right of the reader as shown on the next page.
BioTek Instruments, Inc.
Cleaning the Internal Components |
145
Figure 28: Set the cover upside down and to the right of the reader’s base
Cleaning the Optical Probe(s)
For models without the dispenser module, the optical probes should be cleaned annually. For
models with the dispenser module, the probes should be cleaned at least quarterly. The probes
should also be cleaned if reagent has spilled and/or if an unusually high background signal
has been flagged by the assay controls (typically blanks or negative controls).
Contaminated probes can lead to a loss of sensitivity (e.g., instead of being able to meet the 10
pg/ml concentration detection limit, the instrument may only be able to meet 20 pg/ml).
Another indicator is the %CV in the Corners liquid test—it may increase due to the “Noise” in
the chamber from any spilled fluorescing compounds.
•
To access the optical probes, the first step is to unplug the reader and remove its cover.
If you haven’t already done this, turn to page 144 now for instructions.
•
See page 143 for a list of required materials.
To clean the optical probe(s):
1.
Prepare a small container of isopropyl alcohol.
2.
Identify the probe block inside the reader. Note: The photos on the next page represent
models with the dispenser module and show the internal dispense tube leading from
the tubing port in the rear of the reader to the injector head on the probe block.
3.
Use a screwdriver to remove two screws holding the top optical probe cable in place (see
the photo on the next page).
FLx800 Operator’s Manual
146 | Chapter 6: Preventive Maintenance
Remove
screw
Internal
dispense tube
Remove
screw
Top optical
probe cable
Injector head
Probe block
Figure 29: Removing the two screws holding the top optical probe cable
to the probe block. Model with reagent injection capability shown.
Caution! To avoid damaging the probe, do not excessively
bend the fiber optic cable.
4.
Gently pull the optical probe upward to expose it for cleaning.
Figure 30: Pulling the optical probe upward to expose it for cleaning
5.
Soak the probe in the container of isopropyl alcohol for one minute maximum.
BioTek Instruments, Inc.
Cleaning the Internal Components |
6.
Remove the probe from the container. Wipe the lens with lens-cleaning tissue and set
the probe aside.
Clean with
alcohol and
lens paper
Figure 31: Cleaning the top optical probe
7.
147
If the reader is equipped with a bottom probe, use a cotton swab moistened with
alcohol to clean the lens on the instrument surface.
Figure 32: Cleaning the lens on the bottom optical probe
FLx800 Operator’s Manual
148 | Chapter 6: Preventive Maintenance
Removing/Cleaning the Internal Dispense Tube and Injector Head
Applies to models with the external dispenser module only:
Perform these steps to remove and clean the internal dispense tube and injector head:
1.
Locate the tubing port on the inside rear of the reader. Turn the tube’s thumbscrew
counterclockwise and gently pull the tube from the port.
Figure 33: Detaching the internal dispense tube from the tubing port
2.
Locate the other end of the tube, which is connected to the injector head on the probe
block. Turn the tube’s thumbscrew counterclockwise and disconnect the tube from the
injector head.
3.
Turn the injector head counterclockwise and gently pull it out of its socket.
Figure 34: Removing the injector head from the probe block
BioTek Instruments, Inc.
Cleaning the Internal Components |
4.
5.
149
To clean the internal dispense tube:
•
Soak the tube in hot soapy water to soften and dissolve any hardened particles.
•
Flush the tube by holding it vertically under a stream of water from a faucet.
To clean the injector head:
 Do not remove the o-ring from the injector head (see photos below).
•
Gently insert the stylus (PN 2872304) into the injector head pipe to clear any
blockages. (The stylus should be stored in a plastic cylinder affixed to the rear of
the dispenser module.)
•
Stream water from a faucet through the pipe to be sure it is clean. If the water does
not stream out, try soaking the injector head in hot soapy water and then
reinserting the stylus. The injector head can also be put in an autoclave or
ultrasonic device for cleaning.
O-ring;
do not remove
Figure 35: Cleaning the injector head with the stylus
FLx800 Operator’s Manual
150 | Chapter 6: Preventive Maintenance
Cleaning the Internal Instrument Surface
If reagent has spilled inside the instrument, if the tip prime trough has overflowed (models
with the dispenser module), or as a part of routine cleaning, the internal surface of the reader
may be cleaned.
•
To clean the internal surface, purge the dispenser, unplug the reader, and remove its
cover. If you haven’t already done this, turn to page 144 now for instructions.
•
See page 143 for a list of required materials.
To clean the internal instrument surface:
1.
Moisten a clean cotton cloth with isopropyl alcohol. Note: Do not soak the cloth.
2.
Wipe all sides of the plate carrier. Lift the carrier to clean its bottom surface.
3.
Wipe the instrument’s horizontal surface.
Figure 36: Cleaning the internal surface of the reader
4.
Moisten a cloth with deionized water. Wipe all surfaces that were cleaned with the
alcohol.
5.
Use a clean, dry cloth to dry all wet surfaces.
6.
Use compressed air to remove any dust particles.
BioTek Instruments, Inc.
Cleaning the Internal Components |
151
Reassembling the Components
Perform these steps in the order listed to reassemble the components. Refer to the page
numbers shown for further instructions and photos demonstrating the steps.
1.
Models with the external dispense module only, p. 148:
•
Reinstall the injector head into the probe block. Ensure that the injector head is
properly seated; the knurled plastic should sit flush against the probe block’s
surface.
•
Attach one end of the internal dispense tube to the injector head. Do not
overtighten the thumbscrew!
•
Attach the other end of the tube to the tubing port in the rear of the instrument.
2.
Replace the top optical probe cable and two screws, p. 145.
3.
Replace the reader’s cover and four screws, p. 144.
4.
Models with the dispenser module: connect the module to the reader (communication
cable and external dispense tube).
Performance Check
After reassembling the instrument, perform the following to verify that the instrument is
functioning properly:
•
Plug the instrument in and turn it on; allow its run-time system test to complete.
•
Run a System Test and print the report.
•
Models with the dispenser module: Flush/Purge the fluid path, p. 139.
•
Run any required OQ/PQ tests.
FLx800 Operator’s Manual
152 | Chapter 6: Preventive Maintenance
BioTek Instruments, Inc.
Appendix A
Decontamination
This appendix contains procedures for decontaminating all models of
the FLx800.
Purpose ......................................................................... 154
Required Materials .......................................................... 155
Procedure for Models without the Dispenser Module ............ 156
Routine Procedure for Models with the Dispenser Module ..... 157
Clean Exposed Surfaces .............................................. 157
Decontaminate the Fluid Lines ..................................... 158
Rinse the Fluid Lines ................................................... 158
Clean the Internal Tubing and Injector Head .................. 159
Decontaminate/Rinse the Tip Priming Trough and Priming Plate
................................................................................ 159
Alternate Procedure for Models with the Dispenser Module ... 159
154 | Appendix A: Decontamination
Purpose
Any laboratory instrument that has been used for research or clinical analysis is considered a
biohazard and requires decontamination prior to handling. Intact skin is generally considered
an effective barrier against infectious organisms; however, small abrasions and cuts may not
always be visible.
Decontamination minimizes the risk to all who come in contact with the instrument during
shipping, handling, and servicing. Decontamination is required by the U.S. Department of
Transportation regulations.
Persons performing the decontamination process must be familiar with the basic setup and
operation of the instrument.

BioTek Instruments, Inc. recommends the use of the following
decontamination solutions and methods based on our
knowledge of the instrument and recommendations of the
Centers for Disease Control and Prevention (CDC). Neither
BioTek nor the CDC assumes any liability for the adequacy of
these solutions and methods. Each laboratory must ensure that
decontamination procedures are adequate for the Biohazard(s)
they handle.
Wear prophylactic gloves when handling contaminated
instruments. Gloved hands should be considered
contaminated at all times; keep gloved hands away from eyes,
mouth, nose, and ears. Eating and drinking while
decontaminating instruments is not advised.
Mucous membranes are considered prime entry routes for
infectious agents. Wear eye protection and a surgical mask
when there is a possibility of aerosol contamination. Intact
skin is generally considered an effective barrier against
infectious organisms; however, small abrasions and cuts may
not always be visible. Wear protective gloves when
performing the decontamination procedure.
BioTek Instruments, Inc.
Required Materials |
Required Materials
For all FLx800 models:
•
Sodium hypochlorite (NaClO, or bleach)
•
70% isopropyl alcohol (as an alternative to bleach)
•
Deionized or distilled water
•
Safety glasses
•
Surgical mask
•
Protective gloves
•
Lab coat
•
Biohazard trash bags
•
125 ml beakers
•
Clean cotton cloths
Additional materials for models with the external dispenser module:
•
Screwdriver
•
Small brush for cleaning the tip priming trough and priming plate
•
(Optional) Mild detergent
FLx800 Operator’s Manual
155
156 | Appendix A: Decontamination
Procedure for Models without the Dispenser Module
The sodium hypochlorite (bleach) solution is caustic; wear gloves
and eye protection when handling the solution.
Do not immerse the instrument, spray it with liquid, or use a
“wet” cloth. Do not allow the cleaning solution to run into the
interior of the instrument. If this happens, contact the BioTek
Service Department.
Do not soak the keypad — this will cause damage.
Warning! Internal Voltage. Turn off and unplug the
instrument for all decontamination and cleaning operations.
1.
Turn off and unplug the instrument.
2.
Prepare an aqueous solution of 0.5% sodium hypochlorite (bleach).
•
3.
Be sure to check the % NaClO of the bleach you are using; this information is
printed on the side of the bottle. Commercial bleach is typically 10% NaClO; if this
is the case, prepare a 1:20 dilution. Household bleach is typically 5% NaClO; if this
is the case prepare a 1:10 dilution.
Moisten a cloth with the bleach solution or alcohol. Do not soak the cloth.
•
Wipe the keypad (do not soak). Wipe again with a clean cloth moistened with
deionized or distilled water. Dry immediately with a clean, dry cloth.
•
Wipe the plate carrier and all exposed instrument surfaces of the instrument with
the solution.
•
Remove the top cover and wipe the top surface of the instrument base, as well as
the inside of the top cover. See Chapter 6, Preventive Maintenance for
instructions for removing the reader’s cover.
4.
Wait 20 minutes. Moisten a cloth with distilled water and wipe all surfaces of the
instrument that have been cleaned with the bleach solution or alcohol.
5.
Use a clean, dry cloth to dry all wet surfaces.
6.
Reassemble the instrument as necessary.
7.
Discard the used gloves and cloths using a Biohazard trash bag and an approved
Biohazard container.
BioTek Instruments, Inc.
Routine Procedure for Models with the Dispenser Module |
157
Routine Procedure for Models with the Dispenser
Module

Perform this Routine Procedure when all systems are
functioning normally on the FLx800 with the external
dispenser module. If you are unable to prime the FLx800
due to a system failure, perform the Alternate Procedure
described on page 159.
If disinfecting with sodium hypochlorite (bleach), be
sure to flush repeatedly with deionized water to ensure that
no bleach is carried over. After disinfecting with sodium
hypochlorite, perform the rinse procedure provided on page
158.
If disinfecting with alcohol, do not immediately prime with
deionized water, because the drying effect of the alcohol is
an important aspect of its disinfectant properties.
Clean Exposed Surfaces
1.
Turn off and unplug the instrument.
2.
Prepare an aqueous solution of 0.5% sodium hypochlorite (bleach).
•
3.
4.
Be sure to check the % NaClO of the bleach you are using; this information is
printed on the side of the bottle. Commercial bleach is typically 10% NaClO; if this
is the case, prepare a 1:20 dilution. Household bleach is typically 5% NaClO; if this
is the case prepare a 1:10 dilution.
Moisten a cloth with the bleach solution or alcohol. Do not soak the cloth.
•
Wipe the keypad (do not soak). Wipe again with a clean cloth moistened with
deionized or distilled water. Dry immediately with a clean, dry cloth.
•
Wipe the plate carrier and all exposed instrument surfaces of the instrument with
the solution.
•
Remove the top cover and wipe the top surface of the instrument base, as well as
the inside of the top cover. See Chapter 6, Preventive Maintenance for
instructions for removing the reader’s cover.
Wait 20 minutes. Moisten a cloth with distilled water and wipe all surfaces of the
instrument that have been cleaned with the bleach solution or alcohol.
FLx800 Operator’s Manual
158 | Appendix A: Decontamination
5.
Use a clean, dry cloth to dry all wet surfaces.
6.
Reassemble the instrument as necessary.
7.
If the dispenser module is installed, detach the outlet tube from the rear panel of the
reader. If it is not installed, attach just the dispenser module’s communication cable to
the reader. Remove the supply bottle and its holder.
8.
Perform the procedures described below through page 158 to decontaminate the fluid
line in the dispenser module, the internal tubing and injector head, and the tip priming
trough and priming plate.
Decontaminate the Fluid Lines
1.
Place a beaker with 20 ml of 0.5% sodium hypochlorite solution or 70% isopropyl
alcohol near the syringe on the dispenser module.
2.
Place the syringe’s inlet tube in the beaker.
3.
If you have not already done so, detach the dispenser module’s outlet tube from the
reader’s rear panel.
4.
Place the end of the outlet tube in an empty beaker and set the beaker on the work
surface.
5.
Using Gen5, run two prime cycles, each with a volume of 5000 µl.
6.
Pause for 20 to 30 minutes to allow the solution to disinfect the tubing.
7.
Remove the inlet tube from the beaker of disinfectant solution.
8.
Run one prime cycle with a volume of 1000 µl, to flush the disinfectant out of the fluid
lines.
9.
Empty the beaker containing the outlet tube. Put the tube back in.
10.
Important! If sodium hypochlorite (bleach) was used, perform Rinse the Fluid Lines
below.
Rinse the Fluid Lines
Perform this procedure only if decontamination was performed using sodium hypochlorite.
1.
Place a beaker containing at least 30 ml of deionized water on the dispenser module.
2.
Place the syringe’s inlet tube in the beaker.
3.
If you have not already done so, place the outlet tubes in an empty beaker.
4.
Using Gen5, run five prime cycles at 5000 µl each, for a total of 25000 ml.
5.
Pause for 10 minutes and then run one prime cycle with 5000 µl. This delay will allow
6.
Empty the beaker containing the outlet tubes.
any residual sodium hypochlorite to diffuse into the solution and be flushed out with
the next prime.
BioTek Instruments, Inc.
Alternate Procedure for Models with the Dispenser Module |
7.
Wipe all surfaces with deionized water.
8.
Discard the used gloves and cloths using a Biohazard trash bag and an approved
Biohazard container.
159
Clean the Internal Tubing and Injector Head
Turn to Chapter 6, Preventive Maintenance and perform the following procedures to
access, remove, and clean the internal tubing and injector head:
Required Materials
Removing the Reader’s Cover
Removing/Cleaning the Internal Dispense Tube and Injector Head
When finished, replace the internal components and the reader’s cover.
Decontaminate/Rinse the Tip Priming Trough and Priming Plate
1.
Remove the tip priming trough from the right rear of the FLx800’s microplate carrier.
2.
Wash the tip priming trough and priming plate in hot, soapy water. Use a small brush or
cloth to clean the corners of the trough and plate.
3.
To decontaminate, soak the trough and plate in a container of 0.5% sodium hypochlorite
solution or 70% isopropyl alcohol for 20 to 30 minutes.
4.
If decontaminating in a bleach solution, remove the trough and plate, and thoroughly
rinse with DI water.
5.
If decontaminating with alcohol, remove the trough and plate and let them air dry.
6.
Discard the used gloves and cloths using a Biohazard trash bag and an approved
Biohazard container.
Alternate Procedure for Models with the Dispenser
Module
If you are unable to prime the FLx800 due to a system failure, decontaminate the instrument
and the dispenser module as follows:
1.
Turn to Chapter 6, Preventive Maintenance and perform the following procedures
to remove the cover and remove/clean the internal tubing and injector head. When
finished, leave the cover off the reader and proceed to step 2 below.
Required Materials
Removing the Reader’s Cover
Removing/Cleaning the Internal Dispense Tube and Injector Head
2.
Prepare an aqueous solution of 0.50% sodium hypochlorite (bleach). As an alternative,
70% isopropyl alcohol may be used if the effects of bleach are a concern.
FLx800 Operator’s Manual
160 | Appendix A: Decontamination
•
Be sure to check the % NaClO of the bleach you are using; this information is
printed on the side of the bottle. Commercial bleach is typically 10% NaClO; if this
is the case, prepare a 1:20 dilution. Household bleach is typically 5% NaClO; if this
is the case, prepare a 1:10 dilution.
3.
Moisten a cloth with the bleach solution or alcohol. Do not soak the cloth.
4.
Use the cloth to wipe:
5.
•
All surfaces of the cover (do not soak the keypad)
•
All surfaces of the plate carrier
•
The instrument’s rear panel
•
The exposed surfaces of the dispenser module, including the syringe valve
Remove the external tubing and the syringe from the dispenser module and soak them
in the bleach or alcohol solution. Wait for 20 minutes.
•
To remove the syringe: Carefully pull down the syringe bracket until it stops.
Remove the metal thumbscrew from underneath the bracket. Unscrew the top of
the syringe from the bottom of the syringe drive. Gently remove the syringe and
store it in its original packaging.
6.
Moisten a cloth with DI or distilled water and wipe all surfaces that have been cleaned
with the bleach solution or alcohol.
7.
Rinse all tubing and the syringe with DI water.
8.
Use a clean, dry cloth to dry all surfaces on the instrument and the dispenser module.
9.
Reassemble the instrument and dispenser module as necessary.
10. Discard the used gloves and cloths using a Biohazard trash bag and an approved
Biohazard container.
BioTek Instruments, Inc.
Appendix B
Troubleshooting & Error
Codes
This appendix provides information for contacting BioTek’s Technical
Assistance Center (TAC), should the FLx800 fail to function properly.
In addition, this appendix lists and describes the possible error codes
that may appear on the instrument’s display or in Gen5.
Troubleshooting & Error Codes ..............................................
Product Support & Service ...........................................
Returning Instruments for Service/Repair ......................
Contacting BioTek for Applications Support ....................
Error Codes ....................................................................
Fatal Errors ...............................................................
General Errors ...........................................................
161
162
162
162
163
163
164
162 | Appendix B: Troubleshooting & Error Codes
Product Support & Service
If your instrument(s) or software ever fails to function perfectly, if you have questions
about how to use or maintain them, or if you need to send an instrument to BioTek for
service or repair, please contact our Technical Assistance Center (TAC).
Our Technical Assistance Center is open from 8:30 AM to 5:30 PM (EST), Monday through
Friday, excluding standard U.S. holidays. You can send a fax or an e-mail any time.
Phone:
(800) 242-4685 or (802) 655-4740
Fax:
(802) 654-0638
E-Mail:
tac@biotek.com
Please be prepared to provide the following information:
•
Your name and company information, along with a daytime phone or fax number,
and/or an e-mail address
•
The product name, model, and serial number
•
The reader’s onboard software configuration information. To see this, start at the
reader main menu and press UTIL > TESTS > CHKSUM
•
Gen5 software version information (Help > About Gen5)
•
For troubleshooting assistance or instruments needing repair, the specific steps
that produce your problem and any error codes displayed on the reader or in
Gen5.
Returning Instruments for Service/Repair
If you need to return an instrument to BioTek for service or repair, please contact the TAC
for a Return Materials Authorization (RMA) number and shipping address. Repackage the
instrument properly (see Chapter 3, Installation), write the RMA number on the
shipping box, and ship to BioTek.
Contacting BioTek for Applications Support
BioTek’s fully equipped Application Laboratory provides our on-staff scientists with the
means to assist you with the integration of our instrumentation and software with your
unique scientific applications. If you are having difficulty with optimizing fluorescence
sensitivity or integrating a unique data reduction transformation, or you are just looking
for a recommendation on an appropriate fluorophore, contact us.
Phone: (888) 451-5171
E-Mail: applications@biotek.com
BioTek Instruments, Inc.
Error Codes |
163
Error Codes
An error code is displayed on the instrument display as a four-digit identifier. The first
character will be 0, 1, 2, 3, or A.
•
“0”, “1”, “2”, or “3” indicates a non-critical error, and the instrument will respond
to keypad input. See General Errors for more information.
•
“A” indicates a more serious error with the memory or processing. In this case,
turn off the instrument. Upon restarting the instrument, you should be able to use
the keypad. See Fatal Errors for more information.
 If an error code is displayed, run a System Self-Test for diagnostic
purposes.
Fatal Errors
Fatal Errors indicate conditions that require immediate attention. If a fatal error is
displayed, contact BioTek’s Technical Assistance Center for further instructions.
0 = carrier motor (x-direction)
5 = display
1 = carrier motor (y-direction)
6 = printer
2 = excitation filter wheel
7 = flash memory
3 = emission filter wheel
8 = probe
4 = software timer
9 = memory manager
Code
Description and Probable Causes
A100
Task control block not available
A200
Read already in progress
A300
Motor not available
This error indicates that a motor is not available, but it does not identify which motor
was requested.
A301
Real-time clock not available
A302
Display device not available
A303
Flash memory device not available
A304
Printer device not available
A400
Failed code checksum test on power-up
A501
5 V logic supply voltage level failure detected
A502
24 V power dropped below safe level
A600
Data flash write timed out
A700
Data flash readback did not match write
A800
Code flash write timed out
A900
Memory allocation heap corrupted
AA02
Fluorescence measurement A/D not operational
AA03
Incubator thermistor measurement A/D not operational
FLx800 Operator’s Manual
164 | Appendix B: Troubleshooting & Error Codes
General Errors
General Errors indicate non-fatal conditions that require attention. See the tables below and on
the following pages.
Code
Description and Probable Causes
0100
Abort error
This error code is returned when the read has been terminated with a hard abort
command. In this case all results obtained are invalid.
0101
Abort error
This error code is returned when the read has been terminated with a soft abort
command. In this case the reader will terminate the read and perform a final FMEA
check so that results obtained prior to receiving the abort can be retained as valid data.
Possible Causes:
• User aborted read from Gen5
• User aborted from another serial interface.
0200
X-axis motor did not find the home opto sensor transition
This error indicates that a motor was not able to move to its “home” position as
registered by feedback from an optical sensor.
Note: This error does not look for the xy sensor. See 2400 for xy sensor.
Probable Causes:
• Dirty X-axis rail where the nylon slider bushings are worn and causing too much
friction, or dirt in roller bearings is causing bearings to jam.
• Defective or broken optical sensor.
• Defective motor controller PCB.
• Carrier front support screws are not adjusted or are worn, causing the carrier to
no longer be level. The support pin is no longer inserting properly into the roller
bearings.
• X-axis PCB is not adequately adjusted to the right, and will not allow the flag to
enter the opto sensor enough to trip the sensor. Loosen the two screws and slide
the PCB to the right and retighten. Run the carrier autocal for fluorescence.
• The top probe is running into the plate carrier. Raise the top probe.
0201
Y-axis motor did not find the home opto sensor transition
This error indicates that a motor was not able to move to its “home” position as
registered by feedback from an optical sensor.
Note: This error does not look for the xy sensor. See 2400 for xy sensor.
Probable Causes:
• Dirty Y-axis rails where the bearings are dirty and worn and causing too much
friction.
• Defective or broken optical sensor.
• Defective motor controller PCB or cable.
BioTek Instruments, Inc.
Error Codes |
165
Code
Description and Probable Causes
0202
EX filter wheel did not home
Probable Causes:
• Filter wheel is not inserted into the EX assembly.
• Obstruction is preventing the filter wheel from moving, or the filter is not clipped
in.
• Gear teeth on the filter wheel are binding with the gear teeth on the motor,
preventing the filter wheel from moving.
• Defective or broken Hall Effect sensor.
• Defective motor controller PCB or cable.
• Bearings causing motors to fail.
0203
EM filter wheel did not home
Probable Causes:
• Filter wheel is not inserted into the EM assembly.
• Obstruction is preventing the filter wheel from moving, or the filter is not clipped
in.
• Gear teeth on the filter wheel are binding with the gear teeth on the motor,
preventing the filter wheel from moving.
• Defective or broken Hall Effect sensor.
• Defective motor controller PCB or cable.
• Bearings causing motors to fail.
0204
Probe changer did not home
This error indicates that a motor was not able to move to its home position as registered
by feedback from an optical sensor.
Probable Causes:
• Defective optical sensor.
• Defective motor, motor controller PCB, or cable.
• Obstruction is preventing movement of the probe changer assembly.
0205
Syringe 0 did not home (also known as the first syringe drive)
This error indicates that a motor was not able to move to its home position as registered
by feedback from an optical sensor.
Probable Causes:
• Linear way is dirty or not lubricated, causing the bearings to jam.
• Lead screw is no longer glued to the motor shaft.
• Defective optical sensor.
• Defective motor, motor controller PCB, or cable.
• Cable between the syringe box and the FLx800 is defective, too long, or the
connection has been lost.
0300
Saturation transition failed in the X-axis movement (light beam never found)
This error indicates that during the X-axis movement, the light beam (saturation)
transition (max light to no light) was never found during autocalibration.
Probable Causes:
• Dirty rail and nylon bushings or bearings, causing the carrier to jam.
• Autocal jig is not in the carrier, or mirrors are facing in the wrong direction.
FLx800 Operator’s Manual
166 | Appendix B: Troubleshooting & Error Codes
Code
Description and Probable Causes
0301
Saturation transition failed in the Y-axis movement (light beam never found)
This error indicates that during the y-axis movement, the light beam (saturation)
transition (max light to no light) was never found.
Probable Causes:
• Dirty rail, nylon bushings, or bearings.
• Autocal jig is not in the carrier, or mirrors are facing in the wrong direction.
0400
Carrier x-axis failed positional verify
X-axis motor failed to get to the same position when moved a known number of steps
from the home position and back.
Probable Causes:
• Optical trigger flag has moved or is loose.
• Dirty rail, nylon bushings, or bearings.
0401
Carrier y-axis failed positional verify
Motor y-axis failed to get to the same position when moved a known number of steps
from the home position and back.
• Optical trigger flag has moved or is loose.
• Foreign object in the path of the carrier.
• Dirty rail and bearings.
0402
EX filter wheel failed positional verify
Probable Causes:
• The filter cartridge was removed and then reseated when the instrument was
powered up. Running the system test will clear this error.
• Filter wheel is binding against the motor gear.
• Bearings causing motors to fail.
0403
EM filter wheel failed positional verify
Probable Causes:
• The filter cartridge was removed and then reseated when the instrument was
powered up. Running the system test will clear this error.
• Filter wheel is binding against the motor gear.
• Bearings causing motors to fail.
0404
Probe changer failed positional verify
Note: This error is from basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models.
Probable Causes:
• Optical trigger flag has moved or is loose.
• Obstruction is preventing the probe changer assembly from moving.
0405
Syringe 0 failed position verify (also known as the first syringe drive)
Note: This error is from basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error is similar to 2B01 or 2B02 in basecodes
7080207 or 7080209.
Probable Causes:
• Optical trigger flag has moved, is loose, or has intermittently failed.
• Linear way and bearings are dirty or not lubricated.
• Lead screw is no longer glued to the motor shaft.
• Obstruction is preventing the probe changer assembly from moving.
BioTek Instruments, Inc.
Error Codes |
Code
Description and Probable Causes
0500
Light beam saturated (too much light)
PMT Relative Fluorescing Units (RFU) reached FFFF (99999).
This error can indicate one of the following scenarios:
1. During a fluorescence-dispensing read, if after dispensing, the read times out
because the sample has saturated the PMT.
2. During fluorescence autocalibration, the light level did not reduce to more than
half the saturation light level when the carrier was moved away from center.
Probable Causes:
Scenario 1 (fluorescence-dispensing read)
• Incorrect chemistry was dispensed into the well.
Scenario 2 (fluorescence autocalibration)
• The surface of the auto-calibration jig is too reflective. Return the jig to BioTek
for a replacement jig.
• The carrier did not move far enough to block the light beam. Troubleshoot the
carrier movement.
0501
Light beam saturated (too much light)
PMT Relative Fluorescing Units reached FFFF (99999).
This error can indicate one of the following error scenarios:
1. Prior to a fluorescence read or during a system test, the PMT is tested for
operation. The gain is set at 153. Is the PMT operating properly?
2. PMT is constantly being checked for an overload condition.
Probable Causes:
Scenarios 1 and 2 (fluorescence or system test)
• PMT is defective.
• Connector from the PMT base to the analog board is defective. The
ground/shield is not grounding properly.
• PMT base is defective.
• PMT analog PCB is defective.
0502
Light beam saturated (too much light)
PMT Relative Fluorescing Units reached FFFF (99999).
This error can indicate the following scenario:
Prior to fluorescence read or during a system test, the PMT is tested for operation.
The gain is set at 102. Is the PMT operating properly?
Probable Causes:
• The PMT is defective.
• The connector from the PMT base to the analog board was not built correctly.
The ground/shield is not grounding properly.
• The PMT base is defective.
• The PMT analog PCB is defective.
0505
PMT measurement saturation error occurred while measuring well
Basecode 7080207 replaced this error with the 4XXX series error.
Probable Causes:
• Chemistry in the well is too bright, or the sensitivity is too high.
FLx800 Operator’s Manual
167
168 | Appendix B: Troubleshooting & Error Codes
Code
Description and Probable Causes
0508
PMT measurement saturation (too much light)
This error indicates that during background tests or prior to a read, the PMT reached
saturation. When at rest, the PMT is charged to 650 volts to maintain stability for the next
read. The FLx800 is constantly testing the PMT when idle. If light reaches the PMT, this
error can occur.
Probable Causes:
• Momentary filter overlap. Fixed with 7080207 ver 1.12
• Light leakage or bright light in the read chamber.
• Fluorescence analog PCB has intermittently failed.
0600
Filter gain out of range (failure if PMT saturation is not met within 3 ms)
This error indicates that during fluorescence autocalibration, the gain was increased to
an unsafe level, and the PMT analog PCB did not reach saturation.
Probable Causes:
• The autocalibration jig is incorrectly placed in the carrier. The mirrors are not
facing in the correct direction.
• The EM autocalibration jig is not connected to the PMT analog PCB.
• The fluorescence lamp is not on. 7080207 ver 1.13 ensures the lamp is on at the
beginning of autocalibration.
0601
EX filter position 1 light signal to low
This error indicates that when the EX filter moved to position 1 the light level was too low
at the PMT.
Probable Causes:
• EM filter wheel is not set up correctly. Filter plug is lined up with the light path.
• EX filter position 1 is not setup correctly. Position 1 has a plug.
• PMT or PMT analog PCB is defective.
• The reader could not determine the sensitivity setting using the Autosensitivity
function. Change the Autosensitivity parameters, or adjust the sensitivity setting
manually.
0602
EX filter position 2 light signal to low
This error indicates that when the EX filter moved to position 2 the light level was too low
at the PMT.
Probable Causes:
• EM filter wheel is not set up correctly. Filter plug is lined up with the light path.
• EX filter position 2 is not setup correctly. Position 2 has a plug.
• PMT or PMT analog PCB is defective.
0603
EX filter position 3 light signal to low
This error indicates that when the EX filter moved to position 3 the light level was too low
at the PMT.
Probable Causes:
• EM filter wheel is not set up correctly. Filter plug is lined up with the light path.
• EX filter position 3 is not setup correctly. Position 3 has a plug.
• PMT or PMT analog PCB is defective.
BioTek Instruments, Inc.
Error Codes |
169
Code
Description and Probable Causes
0604
EX filter position 4 light signal to low
This error indicates that when the EX filter moved to position 4 the light level was too low
at the PMT.
Probable Causes:
• EM filter wheel is not set up correctly. Filter plug is lined up with the light path.
• EX filter position 4 is not setup correctly. Position 4 has a plug.
• PMT or PMT analog PCB is defective.
0700
PMT bias current offset
See “Bias current offset” on self-test. Fail if > 5 counts.
This error indicates that the PMT analog PCB noise level is elevated and is not able to
compensate. This is not affected by the PMT or PMT base. The PMT gain is set to 0.
Probable Causes:
• PMT analog board failed to compensate for elevated noise.
• Motor power supply PCB and PMT analog PCB are not properly grounded (poor
connection).
• Motor power supply PCB is noisy, causing PMT analog PCB to be noisy.
• Delay for current to bleed off is to short. 7080207 ver 1.14 increased the delay to
ensure the current has disapated.
0800
Reader failed noise offset test
This error indicates significant variations in background electronic noise were detected
when blocking the light and increasing the gain to maximum. The background noise was
< 700 or > 2450.
Probable Causes:
• Electrical noise may be penetrating the measurement chamber. The bottom and
top shrouds are part of the electrical shielding. Verify that the shrouds are
installed and properly fastened.
• There may be an ambient light leak. Ensure the plate carrier door, the top
optical probe access cover, and the front-hinged door are all properly closed.
• A missing or loose filter may be allowing unintended lamp light into the
measurement chamber. Check the filter wheels for unintended empty positions.
• Analog PCB or faulty internal grounding may be causing internal electronic
noise, or the motor power supply PCB is defective, or both.
• Too much light has saturated the PMT. Turn the unit off and wait 24 hours.
• PMT or PMT base is defective.
• PMT base cable lost ground connection. Rebuild the connector.
• PMT or PMT base is defective or too quiet.
0B00
Invalid <assay number>
Invalid assay selected. Either assay does not exist or number of assays has exceeded //
limit, where “xx” denotes the hexadecimal equivalent of the failed assay number. The
basecode software and/or assays may need to be re-downloaded. Contact BioTek TAC.
0C00
Printer timed out
This error indicates that the time allotted for the instrument to make a valid connection
to a printer has expired.
•
Ensure the printer is connected to the instrument’s parallel port, and is turned
on and full of paper.
0D00
Failed calibration checksum test
FLx800 Operator’s Manual
170 | Appendix B: Troubleshooting & Error Codes
Code
0E01 –
0E04
Description and Probable Causes
Wavelength not found in table
fluorescence
This error indicates that the specified wavelength is not detected in the instrument’s
filter table. The last number is the filter set number in the assay protocol.
Probable Causes:
•
Wavelength or band-pass was not entered correctly or was missing in filter
table.
•
Wavelength or band-pass was entered correctly in the PC software but was
never sent to the reader.
•
Verify that the fluorescence filter table has the wavelengths loaded into the
instrument from the controlling PC software. Compare the contents of the
excitation and emission filter wheels with the software’s filter table.
1000
Necessary configuration data missing
The basecode software and/or assays may need to be re-downloaded.
Contact BioTek TAC.
1100
Failed configuration checksum test
This error indicates that during self-test or at the end of a plate read, the checksums
calculated for configuration flash memory page 0 do not match the saved checksums.
Probable Causes:
• The flash memory on the 7080400 PCB is defective or corrupt. The basecode
software may need to be re-downloaded.
1101
Failed configuration checksum test
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error is similar to 1100 in basecodes 7080207
or 7080209.
This error indicates that during self-test or at the end of a plate read, the checksums
calculated for configuration flash memory page 1 don’t match the saved checksum.
Probable Causes:
• The flash memory on the 7080400 PCB is defective or corrupt. The basecode
software may need to be re-downloaded.
1200
Autocalibration data missing for the fluorescence top/bottom
This error indicates there is no autocal data for either of the two read locations (bottom
probe or top probe).
Probable Causes:
• The 7080400 PCB was changed and the Flash memory does not have the
calibration values loaded. Performing the autocalibration procedure will correct
this.
BioTek Instruments, Inc.
Error Codes |
Code
Description and Probable Causes
13011303
<Motor> not homed successfully
This error indicates that the <motor> has not been homed properly. At the beginning of
the motor_position function, the basecode checks to see if the motor has been homed.
If has not been homed properly, an error is displayed.
Probable Causes:
• If an error 0200 is ignored, see the probable causes for 0200.
Error
Motor
See Probable Causes for
1300*
X-axis
0200
1301
Y-axis
0201
1302
EX motor
0202
1303
EM motor
0203
1304*
Probe changer
0207
1305*
Syringe 1
0208
* In Basecode 7080201 only. Update Basecode to
7080207 or 7080209 for TI models.
1400
1501 –
150F
Assay expects incubation when not supported by instrument
Incubator zone failed temperature range
This error indicates that one or more of the incubator zones failed to maintain their
temperature. The temperature could be too high or too low. After turning the incubator
on, wait at least 10 minutes for the incubator to stabilize.
Zone encoding is as follows:
Zone 1 = 1, 2 = 2, and 3 = 4 (see table below for more information).
Error code
Zone
1501
Zone 1
1502
Zone 2
1503
Zones 1 and 2
1504
Zone 3
1505
Zones 1 and 3
1506
Zones 2 and 3
1507
Zones 1, 2, and 3
Probable Causes:
• Thermistor is defective for that zone.
• Heating pad is defective.
• Motor power supply PCB is defective.
• Incubation chamber is less than 12°C.
FLx800 Operator’s Manual
171
172 | Appendix B: Troubleshooting & Error Codes
Code
Description and Probable Causes
1511 –
1517
Incubator thermistor failed
This error indicates that one or more of the incubator zone thermistors are defective.
After turning the incubator on, wait at least 10 minutes for the incubator to stabilize.
Zone encoding is as follows:
Zone 1 = 1, 2 = 2, and 3 = 4 (see table below for more information).
Error code
Zone
1511
Zone 1
1512
Zone 2
1513
Zones 1 and 2
1514
Zone 3
1515
Zones 1 and 3
1516
Zones 2 and 3
1517
Zones 1, 2, and 3
Probable Causes:
• Thermistor is defective for that zone.
• Motor power supply PCB is defective.
1521 –
1527
Incubator A to D failed
This error indicates that one or more of the incubator zones A to D are defective. After
turning the incubator on wait at least 10 minutes for the incubator to stabilize.
Zone encoding is as follows:
Zone 1 = 1, 2 = 2, and 3 = 4 (see table below for more information).
Error code
Zone
1521
Zone 1
1522
Zone 2
1523
Zones 1 and 2
1524
Zone 3
1525
Zones 1 and 3
1526
Zones 2 and 3
1527
Zones 1, 2, and 3
Probable Causes:
• A to D is defective for that zone.
• Motor power supply PCB is defective.
1530
Door open, causing Zone 3 thermistor error
BioTek Instruments, Inc.
Error Codes |
173
Code
Description and Probable Causes
1540
Incubator Configuration data failure
The FLx800 can be configured for two temperature ranges (25 to 50) or (25 to 65) via
assay configuration download. In addition there is a configuration variable that contains
the actual measured max temperature value pertinent to the range selected. The default
values are 50 and 65 deg C respectively. If one chooses to calibrate the temperature
control then the user would change the value to be the actual measured temp of the
incubator when setting the desired temp to the respective max set-point (50 or 65 deg
C). During powerup or while attempting to change this measured max temp value a test
is performed to insure its validity. If the value stored for this measured max temp is
greater than 5 deg C from the nominal of either 50 or 65 deg C then this error code will
be returned.
Probable Causes:
• Value entered greater than 5 deg C from nominal value depending on the
measured max temp value.
• Assay configuration file loaded supports range not compatible with the
measured max temp value.
1600
Computer control assay definition error
This error is used only for software development.
1700
Kinetic interval not correct for selected options
This error can indicate one of the following error scenarios:
1. The kinetic interval in the current assay is too short.
2. The kinetic interval for a fluorescence plate read is impossible for given
parameters.
3. The kinetic interval for a fluorescence kinetic interval is too large (> 99999) to
transmit.
Probable Causes:
• User programming error.
• Increase or decrease the kinetic interval.
1800
Too many kinetic intervals
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
This error indicates that the combination of assay parameters results in more kinetic
reads than supported by the software.
Change the assay parameters to reduce the number of total kinetic reads.
Note: The FLx800 supports kinetics through computer control only.
1900
Memory allocation failed
This error indicates that during the process of saving data or moving data, the process
failed. If it occurs, however, try turning the instrument off, wait 30 seconds, then turn on
the unit.
Probable Causes:
• The memory is corrupt. Replace the processor PCB.
• If the error persists, contact BioTek TAC.
1A00
Store curve failure
This error indicates a failure during the saving of a standard curve.
FLx800 Operator’s Manual
174 | Appendix B: Troubleshooting & Error Codes
Code
Description and Probable Causes
1B00
Get curve failure
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
This error indicates a failure during the retrieval of a stored standard curve.
1C00
1C01
A/D calibration STBY line never went low
A/D calibration STBY line went low but never transitioned to a high
Note: These errors are from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error is similar to AA02 in basecodes 7080207
or 7080209.
This error indicates a failure with one of the PCBs when trying to initialize the A/D, or
the cable to the defective PCB has lost continuity.
Probable Causes:
• Fluorescence analog PCB or connection between the analog PCB and motor
power supply PCB.
1D00
Results data checksum error
X indicates the read number (or result number up to 8 can be stored in Flash) stored in
flash memory.
1E00
Error in clock communications
This error indicates a problem with the internal clock. Call BioTek TAC.
1F00
1F01 –
1F02
Band-pass overlap in filter set
This error indicates an open hole or overlapping filter set within the optical path of the
excitation and emission filter wheels designated by the protocol. It can also indicate that
the Excitation wavelength is greater than the Emission wavelength.
Equation used for overlapping:
If EM wavelength is > EX
if (EX wavelength + ½ band-pass) >= (EM wavelength – ½ band-pass), then
fail
Otherwise, if EX wavelength is >EM
if (EM wavelength + ½ band-pass) >= (EX wavelength – ½ band-pass), then
fail
Note: 1F01 and 1F02 errors are from Basecode PN 7080201. The unit needs to be
updated to 7080207 or 7080209 for TI models. This error is similar to 1F00 in
basecodes 7080207 or 7080209.
2100
Invalid parameter value selected
This error can occur only during computer control, indicating that an invalid assay
configuration was sent to the instrument.
2200
PMT dark current test signal is < min value for test
This error indicates that the PMT dark current measurment at max gain (1250v)
produces less than 4 counts from PMT.
Probable Causes:
• PMT is defective or missing.
• PMT analog PCB is defective or missing.
BioTek Instruments, Inc.
Error Codes |
175
Code
Description and Probable Causes
2201
PMT test signal too high at the 750 V bias.
See 750 V measurement on self-test. Measured value > 3000 to fail.
This error indicates that the PMT dark current is higher than the specified limit.
Probable Causes:
• The PMT has been saturated due to ambient light leakage.
• The PMT has been damaged.
• PMT base is defective, or the coaxial cable from the base has lost the ground
connection for the shield.
2202
PMT signal too low
Note: In basecode PN 7080207 this test was completely removed. The R4220 PMT
background noise is so quiet that false errors can occur.
2203
PMT 750 V to 500 V ratio test failed defined minimum limit
Note: In basecode PN 7080207 this test was completely removed. The R4220 PMT
background noise is so quiet that false errors can occur.
2204
PMT noise level too high
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
2205
PMT saturation at well during read
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
2301
Lamp voltage failed
This error indicates that the sense resistor monitoring the lamp failed to read the correct
voltage.
Probable Causes:
• Lamp is blown.
• The lamp PCB is defective and needs to be replaced.
2302
Lamp current failed
This error indicates that the lamp is deteriorating, or there is a problem with the lamp
PCB.
Probable Causes:
• Lamp needs to be replaced (filament is shorting together). See “Lamp
Assembly” in Chapter 2.
• Lamp PCB is defective and needs to be replaced.
2303
Lamp is on when it should be off
This error indicates that when checking the lamp status the lamp is on when it should be
off.
Probable Causes:
• Motor controller PCB lamp monitoring circuit failed.
2600
X-Y Limit error
This error can occur only during computer control or autocalibration, indicating that an
invalid assay configuration was sent to the instrument or during the autocalibration
setup the X-Y sensor was not set to No.
27xx
Method doesn’t match first panel assay, where “xx” denotes the failed assay
number
FLx800 Operator’s Manual
176 | Appendix B: Troubleshooting & Error Codes
Code
Description and Probable Causes
28002805
<Motor> currently in use
This error indicates that the <motor> is not available for this model or already has a task
assigned to it. At the beginning of the motor_setup function, the basecode checks to
see if the motor is currently in use or not available.
This error can occur with any motor request; see the table below for errors.
Error
Motor
2800
X-axis
2801
Y-axis
2802
EX motor
2803
EM motor
2804
Probe changer
2805
Syringe 1
Probable Causes:
• User selected the wrong model in the controlling software.
• Incorrect basecode was downloaded.
2B01
Syringe axis motor did not find the home opto sensor transition
This error can indicate one of the following error scenarios:
1. A motor was not able to move to its “home” position as registered by feedback
from an optical sensor.
2. Prior to aspirating, the syringe was not within the homing sensor. To minimize
the air bubble, the syringe must be in the opto sensor.
Probable Causes:
• Linear way is dirty or needs lubrication.
• Defective or broken optical sensor.
• Syringe was not installed correctly or was not cleaned, causing stress on the
syringe movement by not allowing the syringe to move to the home position.
• The glue between the lead screw and the motor has separated.
• Connection between the Dispenser and FLx800 is too long or has intermittently
been lost.
• Syringe valve did not open.
• While moving in the negative direction, the syringe opto sensor triggered off,
then on, indicating that the syringe went past the opto sensor.
2B02
Syringe on sensor when it should be off
This error indicates that if the current position is > 1050 1/16 steps, and the syringe is
on the opto sensor when it should be off.
Note: This error could apply to syringe 1.
Probable Causes:
• Linear way is dirty or needs lubrication.
• Defective or broken optical sensor.
• Syringe was not installed correctly or was not cleaned, causing stress on the
syringe movement by not allowing the syringe to move to the home position.
• Syringe valve did not open.
• The glue between the lead screw and the motor has separated.
BioTek Instruments, Inc.
Error Codes |
177
Code
Description and Probable Causes
2B03
Opto sensor clear count obtained
This error indicates that during syringe initialization, the syringe motor tried to move off
th
the opto sensor and moved > 1050 1/16 steps; did not see the opto transition.
Probable Causes:
• Linear way is dirty or needs lubrication.
• Defective or broken optical sensor.
• Syringe valve did not open.
• Syringe was not installed, was not installed correctly, or was not cleaned,
causing stress on the syringe movement by not allowing the syringe to move off
the home position.
• The glue between the lead screw and the motor has separated.
2B04
Previous syringe move produced an FMEA sensor clear error
This error indicates that the previous move caused the syringe to lose steps.
Probable Causes:
• Linear way is dirty or needs lubrication.
• Defective or broken optical sensor.
• Syringe valve did not open.
• Syringe was not installed correctly or was not cleaned, causing stress on the
syringe movement by not allowing the syringe to move freely.
• The glue between the lead screw and the motor has separated.
2B05
Number of μsteps requested for move too large
This error indicates that during a dispense or move home function, the number of steps
to move was > the maximum syringe travel (944 full steps).
Probable Causes:
• Program or user error.
2C01
Syringe calibration data not set
This error indicates that configuration data for either syringe has not been entered, or
the data is corrupted. This corresponds to the 6 calibration values.
Probable Causes:
• No configuration data was entered.
• Values entered are corrupted or incorrect.
2C02
Syringe calibration checksum failed
This error indicates that the calibration data has been entered, but the checksum failed.
This corresponds to the 6 calibration values.
Probable Causes:
• Values entered are corrupted or incorrect.
2C03
During the validation of the syringe calibration data, the μL/step failed
This error indicates that when the software calculates the μL/step, the results were
either < 0.0100 or > 9.9999.
Probable Causes:
• Values entered are corrupted or incorrect.
2C04
During the validation of the syringe calibration data, the injector position failed
This error indicates that for the requested injector position, the value is not a 0 (bottom
probe) or 1 (top probe). Note: Basecode 7080207 is fixed to the bottom probe only
Probable Causes:
• User attempted to change the injector position to ‘Next to Top Probe’ using
KCjunior.
FLx800 Operator’s Manual
178 | Appendix B: Troubleshooting & Error Codes
Code
Description and Probable Causes
2C05
During the validation of the syringe calibration data, the calibration data failed
This error can indicate one of the following error scenarios:
1. The min calibration value is not = 5.
2. The minimum measured value is not between 2 and 6.
3. The second measured value is not within ± 20% of the calibrated value.
4. The third through fifth measured values are not within ± 10% of the calibrated
value.
5. The calibration values are not in ascending order.
6. The measured calibration values are not in ascending order.
Probable Causes:
• Values entered are corrupted or incorrect.
• Incorrect calibration of the syringe.
2C07
Default calibration data set to “do not use”
This error indicates that the default calibration data flag is set. Syringe needs to be
calibrated.
Probable Causes:
• Factory default data is set; the syringe needs to be calibrated before use or
calibration values need to be entered from syringe drive.
• Values entered are corrupted or incorrect.
2D00
During a dispense read, the reader missed the start of the well read.
This error indicates that during a dispense read, the reader was not able to start the
read for a particular well.
This error can indicate one of the following error scenarios:
1. This error indicates that during a dispense read, the reader was not able to
start the read for a particular well.
2. This error can occur when there is no specific problem with the syringe.
Probable Causes:
• The processor was busy running other tasks.
• A previous task took longer than estimated.
• Software development issue.
2D02
Filter band-pass overlap in intermediate switching position for multi-filter set
reads
This error indicates that the software cannot move either the EX or EM filter wheel
without causing the filters to overlap and saturate the PMT.
Note: Only 2 filter sets can be sent when developing software. The Equation used for
overlapping is:
If EM wavelength is > EX:
if (EX wavelength + ½ bandpass) >= (EM wavelength – ½ bandpass), then fail.
Otherwise, if EX wavelength is >EM:
if (EM wavelength + ½ bandpass) >= (EX wavelength – ½ bandpass), then fail.
Probable Causes:
• The filters placed in the filter wheels are not in the optimum order to prevent this
error.
• The filters defined in the filter table do not match the filter wheels.
• The user failed to send the filter table information to the reader after making
changes to the table.
BioTek Instruments, Inc.
Error Codes |
179
Code
Description and Probable Causes
2D03
EX or EM filters not adjacent for multi-filter set reads
This error indicates either the EX or the EM filter wheels are not set up correctly to have
adjacent filters for a multi-filter set read.
Note: Only 2 filter sets can be sent when developing software.
Probable Causes:
• The filters placed in the filter wheels are not in the optimum order to prevent this
error.
• The filters defined in the filter table do not match the filter wheels.
• The user failed to send the filter table information to the reader after making
changes to the table.
2D04
Filter plug(s) not positioned next to EX filter(s) for light shutter feature during a
dispense / well read
This error indicates that the EX filter wheel is not set up correctly for the light shutter
feature.
Probable Causes:
• The filters placed in the filter wheels are not in the optimum order to prevent this
error.
• The filters defined in the filter table do not match the filter wheels.
• The user failed to send the filter table information to the reader after making
changes to the table.
2D0E
During a dispense read, the reader missed the start of the plate read
This error indicates that during a dispense read, the reader was not able to start the
read. Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
Probable Causes:
• The processor was busy running other tasks.
• A previous task took longer than estimated.
2D26
Dispense kinetic interval out of range
2D27
Time of dispense event overlaps with previous dispense event
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
2D28
Dispenser module not attached
This error indicates that the dispenser is not attached or lost initialization due to
intermittent connection.
Probable Causes:
• Dispenser cable lost connection.
2D29
Dispenser has not been initialized
3100
Plate read took longer than kinetic interval
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
Probable Causes:
• User-defined interval is incorrect.
FLx800 Operator’s Manual
180 | Appendix B: Troubleshooting & Error Codes
Code
Description and Probable Causes
3202
No fluorescence A/D ready transition
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
3203
No incubation A/D ready transition
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
3204
No voltage reference channel A/D ready transition
Note: This error is from Basecode PN 7080201. The unit needs to be updated to
7080207 or 7080209 for TI models. This error does not exist in basecodes 7080207 or
7080209.
PMT overload at well location
This error indicates that at the well specified by the last 3 digits of the error code, the
PMT was saturated.
Probable Causes:
• Chemistry is too bright for sensitivity selected. See “Sensitivity” in Chapter 4 for
tips for selecting the most appropriate sensitivity.
Well location / error code for a 96-well plate.
4000
1
2
3
4
5
6
7
8
9
10
A
4001
4002
4003
4004
4005
4006
4007
4008
4009
B
400D
400E
400F
4010
4011
4012
4013
4014
4015
C
4019
401A
401B
401C
401D
401E
401F
4020
40021
D
4025
4026
4027
4028
4029
402A
402B
402C
402D
E
4031
4032
4033
4034
4035
4036
4037
4038
4039
11
12
400A
400B
400C
4016
4017
4018
40022
4023
4024
402E
402F
4030
403A
403B
403C
F
403D
403E
403F
4040
4041
4042
4043
4044
4045
4046
4047
4048
G
4049
404A
404B
404C
404D
404E
404F
4050
4051
4052
4053
4054
H
4055
4056
4057
4058
4059
405A
405B
405C
405D
405E
405F
4060
Well location / error code for a 384-well plate. Add 40 to the code in the box. If the code
in the box is > FF, add 4 to the code.
1
2
3
4
5
6
7
8
9
A
01
02
03
04
05
06
07
08
09
B
19
1A
1B
1C
1D
1E
1F
20
21
22
23
24
25
26
27
28
29
2A
2B
2C
2D
2E
2F
30
C
31
32
33
34
35
36
37
38
39
3A
3B
3C
3D
3E
3F
40
41
42
43
44
45
46
47
48
D
49
4A
4B
4C
4D
4E
4F
50
51
52
53
54
55
56
57
58
59
5A
5B
5C
5D
5E
5F
60
E
61
62
63
64
65
66
67
68
69
6A
6B
6C
6D
6E
6F
70
71
72
73
74
75
76
77
78
F
79
7A
7B
7C
7D
7E
7F
80
81
82
83
84
85
86
87
88
89
8A
8B
8C
8D
8E
8F
90
G
91
92
93
94
95
96
97
98
99
9A
9B
9C
9D
9E
9F
A0
A1
A2
A3
A4
A5
A6
A7
A8
H
A9
AA AB AC AD AE AF
B0
B1
B2
B3
B4
B5
B6
B7
B8
B9
BA
BB
BC
BD
BE
BF
C0
I
C1
C2
C8
C9
CA
CB
CC
CD
CE
CF
D0
D1
D2
D3
D4
D5
D6
D7
D8
J
D9
DA DB DC DD DE DF
E0
E1
E2
E3
E4
E5
E6
E7
E8
E9
EA
EB
EC
ED
EE
EF
F0
K
F1
F2
F8
F9
FA
FB
FC
FD
FE
FF
100
101
102
103
104
105
106
107
108
114
115
116
117
118
119
11A 11B
11C 11D 11E
11F
120
12C 12D 12E
12F
130
131
132
134
137
138
C3
F3
C4
F4
C5
F5
C6
F6
C7
F7
10
0A
11
0B
113
12
0C
13
0D
14
0E
15
0F
16
10
17
11
18
12
19
13
20
14
21
15
22
16
23
17
24
18
I
109 10A 10B 10C 10D 10E 10F 110 111 112
M
121 122 123 124 125 126 127 128 129 12A 12B
N
139 13A 13B 13C 13D 13E 13F 140 141 142
146
147
148
149
14A 14B
14C 14D 14E
14F
150
O
151 152 153 154 155 156 157 158 159 15A 15B
15C 15D 15E
15F
160
161
162
164
166
167
168
P
169 16A 16B 16C 16D 16E 16F 170 171 172
174
177
178
179
17A 17B
17C 17D 17E
17F
180
143
173
144
145
175
176
133
163
135
165
136
BioTek Instruments, Inc.
Appendix C
Sample Reports
When an assay is run via the reader’s front panel, reports are automatically generated after a
plate has been read (see Specifying Data Output and Reporting Options in Chapter 4 for
information on selecting report formats). In addition, Map, Assay, and Assay List reports can
be printed through the REPORT option from the Main Menu.
This chapter contains samples of the different kinds of reports available on the FLx800.
The report header contains some important information for identifying the assay:
Assay:
After the assay name, the type of assay is identified as (F) or (L), and if
multiple filtersets were defined, the number is also identified here, resembling
(F1) or (L3).
Sensitivity: This value represents either the sensitivity value specified in the assay, or the
result
of the automatic sensitivity selection process.
Filters:
If the METHOD option Wavelength is set to Single, one set of
Excitation/Emission filters is reported here. If the option is set to Dual, both
sets of Excitation/Emission filters are reported here.
Additional Notes:
•
CalcRU is a “generic” term used to represent either the calculated Relative
•
For assays generated through the on-board software: If multiple filtersets are defined
and the METHOD option Wavelength is set to Single, one set of reports is printed for
each filterset. If the Wavelength option is set to Dual, one set of reports is printed,
representing the difference in measurements between filterset #1 and filterset #2.
Fluorescence Unit or the calculated Relative Luminescence Unit, depending on the
assay type.
182 | Appendix C: Sample Reports
Figure 37: Matrix Report, showing the calculated relative fluorescence value (CalcRU), well ID, and
calculated concentration (RSLT) for each well.
BioTek Instruments, Inc.
Sample Reports |
Figure 38: Column Report, showing the plate data for all wells, including
standard deviation and CV% for the standard and sample groups.
ELx808 Operator’s Manual
183
184 | Appendix C: Sample Reports
Figure 39: Curve Report, showing the curve method, equation,
coefficients, and r-square value.
BioTek Instruments, Inc.
Sample Reports |
Figure 40: Matrix Report, showing results for filterset #2 of a Fluorescence assay (F2).
ELx808 Operator’s Manual
185
186 | Appendix C: Sample Reports
Figure 41: Matrix Report, showing results for the difference in measurements between filterset #1 and
filterset #2. In this assay, the METHOD option Wavelengths was set to DUAL.
BioTek Instruments, Inc.
Sample Reports |
Figure 42: Assay Report.
ELx808 Operator’s Manual
187
188 | Appendix C: Sample Reports
BioTek Instruments, Inc.
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