KC4 Manual Update
•
KC4
Manual Update
PN 5281001 Rev C1
KC4 v 2.7 User’s Manual
Congratulations on your purchase of KC4 – a powerful and flexible tool for all of your data collection
and manipulation requirements!
KC4 v2.7 provides new features and enhancements, including:
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Wizard for easily creating Protocols (See page 1)
Customizable toolbar and new icons for most common functions
Interface to the NEW FLx800 Fluorescence Microplate Reader (See page 5)
Monitor Well capability for endpoint and kinetic measurements (See page 12)
Linear Scanning for the PowerWave and ELx808 readers (See page 15)
Automatic Bar Code identification for readers equipped with internal barcode scanners (See page 18)
Kinetic Lag Time calculation for specific applications (See page 20)
Currently KC4's extensive on-board Help system does not support these new features, therefore it is
important that the user thoroughly reviews and keeps handy this Addendum to the User’s Guide. It is
best to review some features while viewing them in the KC4 v2.7 software on-screen.
Protocol Wizard
The Protocol Wizard can be launched from its icon in the toolbar or from the Protocol pull down menu.
It is composed of several tabbed panels, as shown below:
The current panel changes when the user selects another one or when the "< Back" or the "Next >"
buttons are chosen. Typically, when the user creates a new protocol a new protocol is created, the tabs
help ‘step’ through all of the available options within the protocol, beginning with the Protocol Type
panel.
KC4 v 2.7
1
Protocol Type Panel
To create a new protocol, the user must first choose the type of analysis to be defined. When the user
selects a protocol type and moves on to another tabbed panel, the protocol type is ‘set’. The next time the
user accesses the Protocol Type panel, KC4 displays its ‘read only’ state. Only the ‘active’ Protocol
type is visible. The others are grayed, as shown below:
It is possible to begin creating a protocol using the Wizard, and to save it without completing all the
steps in the Wizard. When re-opening the saved protocol to complete the protocol definition, KC4
activates the Plate Settings tab. To create an entirely new protocol, simply click on the New Protocol
button.
Other Protocol Panels
The Protocol Items are conveniently arranged in groups for easy selection and intuitive setup. The other
panels all work the same way. KC4 displays a check mark (9) in front of an item if the user has already
opened this item (via the Wizard, the menu or the toolbar), has modified the data and pushed the OK
button.
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KC4 v 2.7
Each time a user selects an item, KC4 activates the appropriate selection screens. For example, clicking
on Reading Parameters from the Plate Settings panel opens the following screen:
After making the necessary selections, and pressing OK, the Wizard is automatically restored.
KC4 saves the state of each item (checked or unchecked) within a tabbed panel in the protocol file and
restores them when reloading protocol.
The following screens illustrate the remaining available Wizard tabbed panels:
Basic Analysis
KC4 v 2.7
3
Advanced Analysis
Note: The Advanced Analysis options are always available when creating a protocol via the Wizard. If
the Protocol pull-down menu is used, the Advanced option must be selected to make the options
available.
Outputs
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KC4 v 2.7
FLx800 Fluorescence Microplate Reader
The FLx800 is the latest innovative microplate reader from Bio-Tek. It combines superior sensitivity
with easy-to-use features, like removable filter wheels, top and bottom measurements and simple probe
height adjustment. To select the FLx800, go to System/Readers and double click on the FLx800 name
so that it appears in the Current Reader box.
Reader Selection
Configuration
KC4 checks the reader’s configuration during the reading process. If the reading parameters are not
compatible with the reader’s options, such as top or bottom probe availability, KC4 displays a warning
message and aborts the reading.
Filters/Wavelengths
The Filters/Wavelengths dialog box is not available for the FLx800. To view the filters currently
installed on a connected FLx800, click on the Update Filters button in the Reading Parameters.
KC4 v 2.7
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Reading Parameters
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KC4 v 2.7
Reading Type
The FLx800 is primarily a fluorescence microplate reader, however glow luminescence measurements
are also available by selecting the appropriate radio dial.
When Fluorescence is selected, up to six filter pairs can be selected. (The FLx800 can only physically
contain up to four excitation and four emission filters; however, the user may select one or more filter
pairs at different sensitivities or probe orientation).
Update Filters
When the user presses the Update Filters button, KC4 updates the filter drop-down list with the current
filter information stored in the FLx800. If a currently selected filter in the Reading Parameters section
is no longer available on the reader and the user presses the Update Filters button, KC4 displays a
default filter value. If at least one filter has been modified, the following message will be displayed:
At least one of the selected filters is not available anymore and will be replaced by a default filter value.
Plate Type
The FLx800 supports plate types from 6 wells to 384 wells, including Terasaki trays. Area scanning is
not yet available for the FLx800; all plate readings will measure only one point in the center of each
well.
Pre Readings and Monitor Wells
The Pre-Read Blank Plate and Monitor Well options are always available. Neither of these options can
be used in conjunction with the Automatic Sensitivity Adjustment options. Therefore, if the user
selects either the Pre-Read Blank Plate or Monitor Wells, then:
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KC4 disables the Automatic Sensitivity Adjustment options in the Measurement
Options dialog box.
The Automatic Sensitivity Adjustment options of each filter set are automatically
inactivated. In this situation, a warning message is displayed if at least one filter set has
been automatically modified.
Eject between filter sets
The Eject between filter sets option is always available. However, KC4 ignores this option if only one
filter set is selected or enabled. If this option is selected, KC4 ejects the plate and displays
Press OK to start Filter Set … read.
between each filter set reading.
KC4 v 2.7
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Fluorescence Mode
When the Fluorescence reading method is selected, the following features are available:
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Filters: Pull-down lists are available for both excitation and emission filters.
Optics Position: Top is the default position. The user selects Top or Bottom probe reading
position via the drop-down list displayed. If the FLx800 reader attached has only one probe
and an invalid probe is selected, KC4 displays a warning when a plate reading is attempted.
Sensitivity: Valid settings are 0 and integers from 25 to 255. Auto indicates that the Auto
Sensitivity Adjustment feature is enabled. The signal from the range of fluorescence assays
can range from very strong to very weak. The sensitivity of the photomultiplier (PMT) can
be adjusted for a particular assay to ensure that the signals from all wells fall within the
appropriate dynamic range of 0 to 99,999 fluorescence or luminescence units (F.U. or L.U.).
Luminescence Mode
When the Luminescence reading method is selected, the following features are available.
¾ Filters: For the best sensitivity there should be an Open space on the emission wheel. KC4
indicates the Open position as Lum/E. It is possible to select a filter in the emission wheel.
¾ Optics Position: Top is the default position. Bottom probe reading may be selected, however
the signal may be reduced. If the FLx800 reader attached has only one probe and an invalid
probe is selected, KC4 displays a warning when attempting a plate reading.
¾ Sensitivity: Valid settings are 0 and integers from 25 to 255. Auto indicates that the Auto
Sensitivity Adjustment feature has been enabled. The user can adjust the sensitivity of the
photomultiplier (PMT) by increasing the sensitivity value for a particular assay. This ensures
that the signals from all wells fall within the appropriate dynamic range of 0 to 99,999
fluorescence or luminescence units (F.U. or L.U.).
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KC4 v 2.7
In both Fluorescence and Luminescence Mode, there are options available for each filter set. Please
review the KC4 Help System Measurement Options – FL600 for detailed information.
Auto-Sensitivity
There are three options for Auto-Sensitivity Adjustment:
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Scale to High Wells: This method searches for two consecutive gain values where the measured
values are lower and higher than the requested high value wells. The recommended high value
entered is less than 90,000. The High Wells are typically wells with the expected highest signal
on the microplate, such as a high standard.
Scale to Low Wells: This method searches for gain values where the measured values are
lower and higher than the requested low value wells. Low wells would typically be blank wells
or low standards.
KC4 v 2.7
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Best Signal to Noise Ratio: In this method, the user selects Low, High and Experimental
Wells. The Experimental Wells are typically low standards of positive controls. KC4 reads the
wells and searches for the Best Signal to Noise Ratio, without exceeding the High Value. Please
see the KC4 Help System, Automatic Sensitivity Adjustment for more detailed information.
If the user selects more than one filter set, he/she can optionally use the first filter set’s sensitivity for
the second through sixth filter sets sensitivities, as shown below.
Error Messages with the FLx800
KC4 displays errors caused by several. These are shown and explained below.
The user may not select Auto-Sensitivity when Monitor Well is enabled in the reading parameters.
Sensitivity values must be manually set.
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KC4 v 2.7
It is not possible to select Auto-Sensitivity when the Pre-Read Blank Plate option is selected in
Reading Parameters. Users must manually set a sensitivity value.
KC4 protects from overlapping bandpasses according to the following formula:
(emission center wavelength-excitation center wavelength)>((excitation bandpass + emission bandpass)/ 2)
KC4 also protects from bandpasses being too close, and potentially causing overlap according to the
following formula:
(emission center wavelength-excitation center wavelength)>(maximum bandpass+(minimum bandpass/2))
There are also messages that may be displayed during the reading process:
“Top/Bottom probe not installed on the reader”
This message will appear if the user has selected a probe that is not physically present on the connected
FLx800.
“Plug not found in reader filter table”
This message will appear if the read mode is Luminescence and if there is not a plug present in the
Excitation wheel.
“Error (xxx) detected, refer to reader’s Operator’s Manual”
This message will appear if KC4 receives an error message from the reader.
“Max Sensitivity value has been reached”
This message will be displayed if an inappropriate value for the “low well” has been selected and Scale to Low
Wells is chosen in the Auto Sensitivity adjustment. For example, if well A1 is a very low concentration of
analyte, but the user enters a value of 90,000 for an expected well fluorescence or luminescence. In this case, the
Auto Sensitivity adjustment will reach its maximum of 255 before the “low well” has met its expected value. It is
best to enter a low value which closer to 100.
“Min Sensitivity value has been reached”
This message will be displayed if an inappropriate value has been entered and Scale to High Wells is chosen in
the Auto Sensitivity adjustment. For example, if well H1 is a very high concentration of analyte, but the user
enters a “high value” of 1 for the expected value for fluorescence or luminescence. In this case, the Auto
Sensitivity adjustment will reach the minimum gain of 25 and no reading will be taken. It is best to enter a high
value which is closer to 90,000.
KC4 v 2.7
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Monitor Wells
The Monitor Well feature is available for these readers:
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ELx800 (all models)
ELx808 (all models)
PowerWave (all models)
µQuant (all models)
FLx800 (all models)
FL600 (all models)
The Monitor Well feature allows the user to select a well or group of wells to be monitored prior to the
usual plate reading. Monitor Well is available for endpoint and kinetic read modes. Each Reading
Parameter screen contains a checkbox to select this option:
When this is selected, the user then has several options for monitoring wells:
Available Data to monitor depends on the current filter selection of the reading parameters. Only
measured raw absorbance, fluorescence or luminescence values are available for Data to monitor.
Wells to monitor must be contiguous wells, with well positions such as C1-C3 selected.
Stop Monitoring when provides three conditions for the monitoring process to be concluded:
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KC4 v 2.7
Valid operators are >, >=, <, <=.
The user must enter a value to be met by the monitoring criteria.
The Delay between readings selection allows the user to set a delay between each reading of the
monitored wells. The default setting of 00:00:00 allows the reader to read the monitored wells as quickly
as possible.
During the reading process, the following screen is displayed:
Pressing the Read Plate button interrupts the monitored read.
Temperature Controlled Protocols have the following display:
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Shaking
If the user selects Shaking, KC4 will apply it to both the Monitor Well reading process as well as the
Read Sample plate process. If Shake before every read is selected, the plate is shaken before every
reading of the Monitoring process and the Read Sample process. If Shake before first read is
selected, the plate is shaken once before the first read of the Monitor Well process, but not shaken at all
during the Read Sample plate process.
Data Output
The following information may be exported:
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Monitored Wells
Monitor Well Filter Set
Monitor Well Calculation
Monitor Well Limit
None of the Monitor Well data is available for reports.
Error Messages
When the user selects Reading Parameters for the FLx800 and FL600, the following message may
appear:
Automatic Sensitivity Adjustment is not allowed when the Monitor Well option is selected. Automatic
Sensitivity Adjustment will be disabled.
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KC4 v 2.7
Linear Scanning
Linear Scanning across a well is available on all ELx808 and PowerWave readers only. The linear
scanning function allows the user to select multiple points across a well in order to observe a pattern
which may be present in the well bottom, such as an agglutination pattern. If a PowerWaveX Select is the
connected reader, scanning is available only when the 96 well plate type is selected.
Reading Parameters
A Scanning button has been added to the Reading Parameters screen:
When the user presses the Scanning button, the dialog prompts him/her to select the total number of
points to be read across each well. The range is between 1 and 31 and must be an odd number.
Linear scanning is available only in the endpoint reading mode, if more than one scanning point is
selected. The default setting is one point per well for typical endpoint, kinetic and spectrum scanning
(PowerWave readers only).
Linear scanning is available only for single wavelength readings. If blank wells are defined, the blanks
are not automatically subtracted. Transformations may be performed only on calculated data, not on the
individual scanned data points.
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Calculations
The following analyses are available for the scanned data points:
The integral calculation option will provide an area under the curve according to the trapezoidal method,
shown below:
i = n −1
where Y= absorbance and
yi + yi +1 
Integral= 
×(xi +1− xi)

X= scan point value
2
∑
i =1

Note that in linear scanning, (xi +1− xi) is equal to 1, and thus, the calculation formula is reduced to:
i=n
y1+ yn 
Integral=∑ yi −

 2 
i =1
Measurement Options
For each of the possible calculations available, the user may select the appropriate number of points to
be used in the calculation. This option is selectable in the Calculation Zone dialog boxes.
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KC4 v 2.7
Data Results View
Immediately after the plate is read, the scanning curves are displayed:
The user is able to Zoom in on selected wells by moving the cursor to the appropriate well position and
clicking on the well. Several wells may be overlaid in a zoomed screen. Press the Ctrl key while
selecting up to 8 wells to overlay.
Reporting and Exporting
The following data is available for the printed report:
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Scanning curves
Well zoom
Table of scanned points
The table of scanned points is available for exporting.
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Barcode Identification
This feature allows the barcode information from each plate to be automatically entered into KC4. This
feature is available only for the ELx808 and PowerWave readers, if equipped with an internal barcode
scanner.
A checkbox is available on the Reading Parameters screen. To select for automatic barcode
identification, select this checkbox.
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KC4 v 2.7
The Read Barcode button is available even if the reader is not equipped with a barcode scanner. If the
user selects the option and the reader does not have a barcode scanner, the following message will
appear:
Identification values may be entered manually. The maximum number of characters is 32.
The Plate Description window displays the barcode information.
It also appears in the Assay Description section of the printed report.
Options
In the Protocol/Options dialog box, the user can select to Use Barcode as Filename option. When this
option is selected, KC4 automatically saves the data file at the end of the read with a name identical to
the barcode ID.
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Kinetic Lag Time
When the user selects Max V in the Kinetic Analysis section, an additional calculation is now available.
The user may obtain the Lag Time calculation, which is defined as the time intercept between the line of
maximum slope of the propagation phase and the absorbance baseline at time = 0. See diagram below.
Absorbance
Point of max
slope
Lag Time
Time (Min)
By default, the calculation zone starts at t = 0.
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KC4 v 2.7
To see the Lag Time within a given well, Zoom on the well by clicking on it while in the kinetic curves
data display.
Report and Export
Lag time is selectable as a component of the report items available, as well as in the export items
available.
KC4 v 2.7
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KC4 v 2.7
BIO-TEK
®
INSTRUMENTS, INC.
KC
4
4
User’s Guide
User's Guide to
KC4
April 2000
Copyright  2000
Bio-Tek Instruments, Inc.
User's Guide PN 5281001, Revision C
ii
Notices
BIO-TEK INSTRUMENTS, INC.
Highland Park, Box 998
Winooski, Vermont
05404-0998 USA
Phone:
(802) 655-4040
(888) 451-5171
(800) 24-BIOTK (Service)
Fax:
(802) 655-7941 (Sales)
(802) 655-3399 (Service)
E-Mail:
[email protected]
Internet Site:
http://www.biotek.com
European Coordination Center
Bio-Tek Instruments, S.a.r.l.
Parc d'Activités de l'Observatoire
43 Avenue des Trois Peuples - Bât E
78180 Montigny-Bretonneux France
Phone:
33 1 39 30 2276
Fax:
33 1 39 30 0987
 Copyright 2000.
No part of this publication may be reproduced, transmitted, transcribed,
stored in a retrieval system, or translated into any language without the
written permission of Bio-Tek Instruments, Inc.
Printed in the United States of America.
Bio-Tek Part Number 5281001.
Trademarks
Bio-Tek is a registered trademark, and KC4 is a trademark of Bio-Tek
Instruments, Inc. Bio-Cell is a trademark of Bio-Tek Instruments and BioCell is patent-pending.
Microsoft is a registered trademark and Windows and Windows 95/NT are
trademarks of Microsoft Corporation.
Restrictions and
Liabilities
Information in this document is subject to change, and does not represent a
commitment by Bio-Tek Instruments, Inc. Changes made to the information
in this document will be incorporated in new editions of the publication. No
responsibility is assumed by Bio-Tek for the use or reliability of software or
equipment that is not supplied by Bio-Tek, or its affiliated dealers.
iii
Limited Software
Warranty
Bio-Tek Instruments, Inc. (“Bio-Tek”) warrants, for a period of ninety (90)
days after the date of delivery, that all software manufactured and sold by
Bio-Tek (the “Software”) will conform, as to all substantial operational
features, to Bio-Tek’s then current published specifications (meaning those
specifications in effect as of the date of purchase) and will be free of defects
which, in Bio-Tek’s sole judgment, substantially affect system performance.
This Warranty is limited to the original purchaser (the “Purchaser”) and
cannot be assigned or transferred. All claims under this Limited Warranty
must be made in writing or fax to Bio-Tek attention: Service Department.
The Purchaser must notify Bio-Tek in writing within ninety (90) days after
the date of delivery of the Software to the Purchaser of any claim under this
Limited Warranty. This 90 day period shall not be extended by the delivery
or receipt of any subsequent modifications to the Software. Modifications to
Software within a version (“Updates”) are expressly excluded from the terms
of this Warranty. If the Software is found to be defective by Bio-Tek,
Bio-Tek’s sole obligation under this Warranty is to use efforts consistent
with Bio-Tek’s regular business practices to attempt to remedy such defect.
In no event shall Bio-Tek’s liability under this warranty exceed the original
purchase price of the Software.
This is a limited warranty. This warranty contains the entire obligation
of Bio-Tek. No other warranties, express, implied, or statutory, are
given. The implied warranties of merchantability and of fitness for any
particular purpose are expressly excluded and do not apply to the
software.
Under no circumstances shall Bio-Tek be liable to the purchaser or any
other person for any direct, incidental, or consequential damages,
whether arising out of breach of warranty or otherwise.
This warranty shall not be changed or modified in any way without the
express written permission of an officer of Bio-Tek Instruments, Inc.
This Warranty shall be NULL AND VOID if any modifications are made to
the Software by the Purchaser. Correction of difficulties or defects traceable
to Purchaser’s errors or system changes shall be billed to Purchaser at
Bio-Tek’s standard time and materials charge.
Licensing Agreement
LICENSE: You have the non-exclusive right to use the enclosed KC4
program. This program can only be used on a single computer, unless a site
license or additional network hardlocks have been purchased through BioTek Instruments. You may transfer the program from one computer to
another. For complete functionality, a KC4 hardlock must be attached to the
controlling computer. You may not electronically transfer the program from
one computer to another. You may not distribute copies of the program(s) or
documentation without the prior written consent of Bio-Tek Instruments,
Inc., or as stipulated in a sub-licensing agreement. You may not use, copy,
modify, or transfer the program or documentation, except as expressly
provided in this License Agreement.
iv
COPYRIGHT: The KC4 program and its documentation are copyrighted.
You may not copy any program or the documentation except as for loading
the program into the computer as part of executing that program and for
back-up purposes, respectively. You must reproduce and include the
copyright notice on the back-up copy.
TERMS: This license is effective until terminated. You may terminate it by
destroying the program(s) and documentation and all copies thereof. This
license will also terminate if you fail to comply with any term or condition
of this Agreement. You agree upon such termination to destroy all copies of
the programs and documentation.
Document
Revision
Revision
Date
Changes
A
October 1998
First issue.
B
October 1998
Remove automatic update of Axis Title fields.
Update note re: “Use barcode as file name”
option not available in KC4 v2.5.
Update informative Reading parameters
screen shot in Appendix B.
General cosmetic improvements.
C
April 2000
Update contact information for European
Coordination Center.
Update throughout to add the Luminescence
reading mode for the FL600.
Update to show support for the PowerWaveX
Select reader.
Update Chapter 6 “Wavelengths” topic to
discuss new Calibrate button.
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Contents
Overview
1-1
KC4.........................................................................................................................................1-1
Concepts ...................................................................................................................1-3
Structure ...................................................................................................................1-3
File Types .................................................................................................................1-3
The KC4 Module ....................................................................................................................1-4
Data Menu ................................................................................................................1-4
Protocol Menu ..........................................................................................................1-5
Display Menu............................................................................................................1-6
System Menu ............................................................................................................1-5
Main Window ...........................................................................................................1-6
The Biograph Module .............................................................................................................1-6
File............................................................................................................................1-6
Graph ........................................................................................................................1-7
Using KC4: Step-by-Step.......................................................................................................1-9
Technical Support .................................................................................................................1-10
Installing the Software
2-1
Read Me First! ........................................................................................................................2-1
System Requirements..............................................................................................................2-2
Installing the Security Key ......................................................................................................2-2
Software Installation ...............................................................................................................2-3
To Install KC4 ..........................................................................................................2-3
Connecting a Reader to the Computer ....................................................................................2-7
Starting KC4 ...........................................................................................................................2-7
Getting Started
3-1
Main Screen ............................................................................................................................3-1
Title Bar....................................................................................................................3-1
Menu Bar ..................................................................................................................3-1
Toolbar .....................................................................................................................3-2
Status Bar..................................................................................................................3-3
Navigating the Help System....................................................................................................3-4
Sample Files............................................................................................................................3-5
Data Menu
4-1
Data Menu Quick Reference ...................................................................................................4-1
Working with Plates................................................................................................................4-3
New Plate..................................................................................................................4-3
User's Guide to KC4
Contents • 1
Open......................................................................................................................... 4-3
Close ........................................................................................................................ 4-4
Save ......................................................................................................................... 4-4
Save As .................................................................................................................... 4-4
Exporting Plate Data .............................................................................................................. 4-5
Export File ............................................................................................................... 4-6
Append to Kinetic File ........................................................................................................... 4-7
Printing Reports ..................................................................................................................... 4-8
Print ......................................................................................................................... 4-8
Print Preview ........................................................................................................... 4-9
Print Setup ............................................................................................................. 4-10
Plate Description .................................................................................................................. 4-11
Samples ................................................................................................................................ 4-12
Import Sample Names............................................................................................ 4-13
Export Sample Names............................................................................................ 4-13
Printing Sample IDs............................................................................................... 4-13
Read Plate ............................................................................................................................ 4-14
Entering Plate Data Manually ................................................................................ 4-15
Reading Plate Data from a File .............................................................................. 4-17
Edit Menu
5-1
Editing Tools.......................................................................................................................... 5-1
Cut ........................................................................................................................... 5-1
Copy......................................................................................................................... 5-1
Paste......................................................................................................................... 5-1
Undo ........................................................................................................................ 5-2
Protocol Menu
6-1
Protocol Menu Quick Reference ............................................................................................ 6-2
Working With Protocols ........................................................................................................ 6-4
New Protocol ........................................................................................................... 6-4
Open Protocol .......................................................................................................... 6-4
Modifying a Protocol ............................................................................................... 6-5
Close Protocol.......................................................................................................... 6-6
Save Protocol / Save As........................................................................................... 6-6
Plate Description .................................................................................................................... 6-7
Defining Reading Parameters................................................................................................. 6-8
Reading Type........................................................................................................... 6-8
Reader Model .......................................................................................................... 6-9
Kinetic Parameters................................................................................................... 6-9
Kinetic Parameters (FL600)................................................................................... 6-10
Filters/Wavelengths ............................................................................................... 6-11
Wavelengths........................................................................................................... 6-12
Plate Options.......................................................................................................... 6-13
Plate Shaking ......................................................................................................... 6-15
Temperature Control.............................................................................................. 6-16
Pre-Readings.......................................................................................................... 6-16
Extended Range ..................................................................................................... 6-18
Filter Sets (FL600)................................................................................................. 6-19
2 • Contents
User's Guide to KC4
Measurement Options (FL600)...............................................................................6-21
General Options (FL600)........................................................................................6-23
Scanning (FL600) ...................................................................................................6-26
Raw Data Correction.............................................................................................................6-28
Blank Plate Subtraction ..........................................................................................6-30
Blank Wells Subtraction .........................................................................................6-30
Pathlength Correction .............................................................................................6-31
Kinetic Analysis ....................................................................................................................6-32
Data to process .......................................................................................................6-32
Mean V ...................................................................................................................6-33
Max V.....................................................................................................................6-33
Mean Min/Max .......................................................................................................6-34
Onset OD / Onset OD %.........................................................................................6-35
Formula...................................................................................................................6-36
No Calculation........................................................................................................6-36
Spectrum Analysis ................................................................................................................6-37
Data to Process .......................................................................................................6-37
Min/Max .................................................................................................................6-37
Formula...................................................................................................................6-38
Plate Layout ..........................................................................................................................6-39
Identifiers................................................................................................................6-39
Dilutions .................................................................................................................6-43
Standards ................................................................................................................6-44
Spikes .....................................................................................................................6-45
Conc./Dil. ...............................................................................................................6-46
Single Plate Transformations ................................................................................................6-47
Formula Syntax.......................................................................................................6-48
Formula Examples ..................................................................................................6-49
Multi Plate Transformations .................................................................................................6-50
Syntax .....................................................................................................................6-51
Automatic Formula Generation ..............................................................................6-51
Curves ......................................................................................................................... ..........6-52
Curve Description...................................................................................................6-52
Curve Axes .............................................................................................................6-53
Curve Fit .................................................................................................................6-55
Curve Interpolation.................................................................................................6-57
Validation..................................................................................................................... .........6-58
Cutoffs ..................................................................................................................................6-60
Reports........................................................................................................................ ..........6-62
Log Report..............................................................................................................6-63
Kinetic Curves Report ............................................................................................6-63
Well Zoom Report ..................................................................................................6-64
Validation Report ...................................................................................................6-64
Cutoffs Report ........................................................................................................6-65
Sample Name List Report.......................................................................................6-65
Plate vs. Table Format ............................................................................................6-65
Statistics Report......................................................................................................6-66
Export ......................................................................................................................... ..........6-67
Protocol Options ............................................................................................................... ....6-68
User's Guide to KC4
Contents • 3
Display Menu
7-1
Display Menu Quick Reference ............................................................................................. 7-1
Curve(s)....................................................................................................................... ........... 7-2
Validation Results .................................................................................................................. 7-3
System Menu
8-1
System Menu Quick Reference .............................................................................................. 8-2
Reader Selection ............................................................................................................... ..... 8-3
FL600 Configuration ............................................................................................... 8-4
Port Selection........................................................................................................... 8-4
Filters/Wavelengths ................................................................................................. 8-5
Filters/Wavelengths (FL600) ................................................................................... 8-6
SuperUser Login .................................................................................................................... 8-8
Change SuperUser Password ................................................................................... 8-8
Define Protected Functions...................................................................................... 8-8
Setup .......................................................................................................................... .......... 8-10
Plate Format ................................................................................................................... ...... 8-12
Microplate Measurement ....................................................................................... 8-14
Reader Control ................................................................................................................. .... 8-15
Door/Carrier........................................................................................................... 8-15
Pre-Heating ............................................................................................................ 8-16
Diagnostics........................................................................................................................... 8-17
Optics Test............................................................................................................. 8-17
Optics Test (FL600)............................................................................................... 8-19
Universal Plate Test ............................................................................................... 8-20
Results
9-1
Overview................................................................................................................................ 9-1
Data........................................................................................................................................ 9-2
Transformation Results ............................................................................................ 9-4
Calculated Concentrations ....................................................................................... 9-4
Concentrations x Dil. ............................................................................................... 9-5
Symbols ................................................................................................................... 9-6
Statistics ................................................................................................................................. 9-7
Viewing Kinetic Data............................................................................................................. 9-8
Viewing Spectral Scan Data................................................................................................... 9-9
Viewing Scanned Data (FL600)........................................................................................... 9-11
Biograph
10-1
Overview.............................................................................................................................. 10-1
File Menu ............................................................................................................................. 10-2
File Menu Quick Reference ................................................................................... 10-2
New........................................................................................................................ 10-3
Save ....................................................................................................................... 10-3
Open....................................................................................................................... 10-3
Save As, Close, Print Preview, Print..................................................................... 10-4
Validate and Update KC4 ...................................................................................... 10-4
4 • Contents
User's Guide to KC4
View Menu............................................................................................................................10-4
View Menu Quick Reference..................................................................................10-4
Graph Menu ..................................................................................................................... .....10-5
Graph Menu Quick Reference ................................................................................10-5
Standards ................................................................................................................10-6
Axes, Fit, Interpolations..........................................................................................10-7
Overlaying Graphs..................................................................................................10-7
Curve Window ......................................................................................................................10-9
Appendix A
11-1
Using KC4 in a Robotic System............................................................................................11-1
User's Guide to KC4
Contents • 5
6 • Contents
User's Guide to KC4
Overview
KC4
KC4 provides rapid, flexible, and powerful assay analysis for myriad
applications. The user interface is simple and uncluttered, providing a quick,
step-by-step setup and read process for even the most complex assays.
Applications include:
•
Research
•
DNA quantitation
•
Fluorescence and luminescence assays
•
Bio-Industrial
•
Pharmaceutical
•
Development
•
Interfaces to all Bio-Tek microplate readers.
•
Defines and controls all reading protocols:
KC4:
¾Selection of filters/wavelengths.
¾Shaking parameters.
¾Incubation.
¾Flexible kinetic options.
¾Optional manual entry of data.
¾Reader diagnostics testing and archiving.
•
User's Guide to KC4
Provides the option to prevent modification of protected
protocols.
Overview • 1-1
•
Allows you to customize assay development:
¾Graphical input for template creation, including autoreplication and fill.
¾Quick entry of standard concentrations and sample dilutions
with “auto-increment” and “auto-factor”.
¾Control and assay validations may be created for each assay.
¾Multiple sample curves displayed and analyzed
simultaneously, including interpolations.
¾Powerful multi-level transformation formulas allow
manipulation of raw data, transformed data, curve-fit results,
or statistical calculations at any point in the analysis.
¾KC4’s “zoom” feature allows detailed information observed
for each well, including the ability to highlight specific
kinetic reaction wells (absorbance and fluorescence).
¾Sample IDs may be manually entered or imported.
¾Extensive curve-fitting capabilities, including Linear or Log
axis definition, Point-to-Point, Linear, Polynomial, Cubic
Spline, 4-P, and interpolation and graphic editing features.
•
Allows you to customize the results display and printed reports:
¾Quick access to all available result data, usually from a
single screen: Measured ODs, transformed ODs, kinetic
calculations, concentrations, template layout, statistical data
on controls, standards, and samples, and a masking and
recalculation capability.
¾Kinetic options: Real-time display of kinetic reactions,
individual well zoom, Mean V, Max V, time at Max V,
Min/Max OD, and Onset calculations.
¾Screening options: Definition of cutoffs by algebraic
formulae, and positive, negative, etc., range calculations.
¾Validation options: Formula to validate the Mean, Standard
Deviation, or CV of controls.
¾Printed reports may be completely customized: The order of
report segments, items that are included within each report
segment, page breaks, matrix or column formats, header and
title options, print preview function.
•
Flexible data exporting and networking:
¾Data may be exported automatically to a number of other
programs for further manipulation or report merging.
¾Exporting is not limited to just raw data -- all calculated
results may be exported.
1-2 • Overview
User's Guide to KC4
Concepts
What is meant by “Data” in KC4?
Data is information attached to a microplate. Data includes:
•
Raw values sent by the reader, either optical density,
fluorescence, or luminescence data values.
•
Sample names which you either import or enter manually.
•
Plate description (date/time, prompts, comments).
What is meant by “Protocol” in KC4?
•
Data acquisition method, end-point, kinetic, spectrum scanning
(reader-dependent), well area scanning (reader-dependent), or
defining reading parameters such as wavelengths, plate shaking,
and temperature control.
•
Calculation method (data reduction) – how the data is evaluated.
•
Reporting and exporting procedures – the presentation content
and format.
Structure
The KC4 software package is composed of the KC4 and Biograph modules.
Windows-style menus, toolbar buttons, and pull-down lists guide you
through the steps involved in defining protocols.
File Types
Plate data files of the type *.PLA contain the following information:
•
Raw data.
•
Plate description (prompts, comments, data, and time of
reading).
•
Sample names.
•
Reading parameters (reader name, filter values, shaking
parameters etc.).
Global data files of the type *.GLB are files containing plate information as
well as descriptive information for the protocol with which the data was
retrieved.
Graph files of the type *.GPH are graphic files containing curve information,
used for archive purposes.
User's Guide to KC4
Overview • 1-3
The KC4 Module
This section describes the full menu system of KC4. Each menu is discussed
in the order in which it appears from left to right across the top of the KC4
main screen.
Details on how to use these menus in the course of creating and evaluating a
plate assay are discussed in later sections.
Data Menu
The Data menu is used to process data associated with a plate, including the
plate description, sample names, and raw data retrieved from the reader.
1-4 • Overview
Menu Item
Description
New Plate
Clear memory and prepare KC4 to accept a new set of
data.
Open
Open a data (*.PLA or *.GLB) file.
Close
Close a data (*.PLA or *.GLB) file.
Save / Save As
Save a data (*.PLA or *.GLB) file.
Export
Export data according to the format defined in the
Protocol.
Append to Kinetic
File…
Append new data to an existing data file.
Print
Print the results data according to the format defined in
the protocol.
Print Preview
View the report formatting on the screen before printing.
Print Setup
Define the printer parameters using the Windows Print
Manager.
Plate Description
Enter comments associated with a particular plate.
Samples…
Identify individual samples. IDs can be manually
entered, automatically assigned, or imported.
Read Plate…
Acquire data automatically from a reader, or acquire
data that was manually entered or imported from a file.
The filing of data enables the storage and retrieval of
information attached to a plate.
Exit
Close KC4 and return to the Windows operating system.
User's Guide to KC4
Protocol Menu
The Protocol menu is used to create and modify KC4 Protocols.
User's Guide to KC4
Menu Item
Description
New Protocol
Select the type of assay protocol to be defined. Options
include: Qualitative, Quantitative, Curve Comparison,
Toxicology, and Endotoxin assays.
Open
Open a protocol (*.PRT) file.
Close
Close a protocol file.
Save / Save As
Save a protocol file.
Plate Description
Define the prompts that appear each time a plate is read.
Reading
Define reading parameters. Parameters include endpoint, kinetic, or spectrum readings, filter/wavelength
choices, shaking, temperature control, lag time before
reading, kinetic time parameters, and scaling of kinetic
graphical display. Available options are reader
dependent.
Raw Data Correction
Enable/Disable corrections to raw data, including Blank
Plate Subtraction, Blank Wells Subtraction, and/or
Pathlength Correction.
Kinetic Analysis
Select the type of data and the method of kinetic data
analysis. Options: Mean V, Max V, Mean Min OD,
Mean Max OD, Onset OD, and Formula.
Spectrum Analysis
Select the type of data and the method of spectrum data
analysis. Options: Min/Max and Formula. A Result
Name can be specified for use as a variable in later
calculations.
Plate Layout
Assign meaningful identifiers to the data by attaching
labels to the wells. Identifiers are protocol-specific.
Dilution options allow automatic or manual filling,
replication, and assigning of concentrations or dilutions
where appropriate.
Single Plate
Transformations
Perform calculations on data from any well within the
plate.
Multi Plate
Transformations
Define up to 3 levels of calculations between different
sets of plate data.
Curves
Select the data type to be plotted and select the curve
fitting routine and graphical display options.
Validation
Define equations to determine whether the results are
within acceptable limits.
Overview • 1-5
Cutoffs
Define cutoff parameters used to categorize data into
groups, identified by symbols. Cutoffs can be formulas
or absolute values.
Report
Define the content and format of the printed report.
Export
Define the content and format of the exported file. This
file can then be used by other applications.
Options
Optionally select the automatic naming and filing of data
files. Other features are the automatic reporting and
exporting of data.
Advanced
The Advanced option provides access to additional
protocol configuration features, such as Plate
Description, Raw Data Correction, Multi-Plate
Transformations, Validation, and Cutoffs.
Recent File
Shows the last 4 protocol files you have opened.
Display Menu
The Display menu is used to check the verification status of data and/or to
activate the Biograph module to view, adjust, and otherwise interact with
test results as displayed on a curve or within a curve set.
Menu Item
Description
Curve(s)
Open the Biograph module to view, modify, or store the
standard curve, and to remove outlying points.
Validation Results
Examine the status of the calculated validation formula.
Toolbar
Show or hide the toolbar.
System Menu
The System menu is used to select or change the current reader, to set up the
reader for use with KC4, and to perform reader diagnostics. In addition, the
KC4 system administrator (the “SuperUser”), can use this menu to set
passwords and certain system parameters.
1-6 • Overview
Menu Item
Description
Readers
Select a reader model and set reader parameters such as
top or bottom probe sizes for the FL600 reader,
communication options, filter/wavelength settings, and
to retrieve or download wavelength settings (readerdependent).
SuperUser Login
Provide the administrator with access to protected
functions.
User's Guide to KC4
Change SuperUser
Password
Define a new SuperUser password.
Define Protected
Functions
(SuperUser) Restrict/permit access to modifying
protocols, modifying values, and masking values.
Setup
Select Import/Export separators, time formats, and
weighting of Standard Deviation.
Plate Formats
Applies only to the FL600. Use this to add a new plate
format, change an existing format, and to select a
default.
Reader Control
Turn incubation on/off, set the temperature, and control
movement of the plate carrier in and out of the plate
chamber.
Diagnostics
Perform an Optics Test or Universal Plate Test. View
historical test results.
Main Window
Once a protocol has been defined and plate data has been acquired, you can
view and manipulate the data with a range of available options.
•
View the raw OD values, Fluorescence Units (FU), or
Luminescence Units (LU), and the calculated results.
•
Mask outlying wells.
•
Change data values.
•
Get statistics on a particular set of data.
•
Zoom in on a particular well (e.g. kinetics or spectral scan).
•
Copy information to the clipboard.
The Biograph Module
Biograph can be loaded from within KC4 or separately using the Biograph
icon. If used in standalone mode, Biograph can generate manual graphic
curves via the main window. It can also generate standard curves from data
presented using KC4.
File
User's Guide to KC4
Menu Item
Description
New
Create a new graphic curve.
Open
Open an existing archived curve.
Close
Close a curve.
Overview • 1-7
Save / Save As
Save a curve.
Print
Print the current graphic.
Print Preview
View the current graphic on the screen before printing.
Print Setup
Define the printer parameters using the Windows Print
Manager.
Recent File
Shows the last 4 graph files opened.
Validate and
Update KC4
Validate changes, close the Biograph module, and bring
updates into KC4.
Exit
Return to the program from which Biograph was
launched (KC4 or Windows operating system).
Graph
1-8 • Overview
Menu Item
Description
Standards
Manually create a new curve. The standard
concentrations and response values are defined via a
spreadsheet-like interface screen. Means and Standard
Deviations are automatically calculated for replicate
response values.
Axes…
Select the x axis transform (Lin / Log), Extrapolation
factor, grid display and axis title. You can select the y
axis transformation (Lin / Log), axis title, display grid,
and axis range. The graphic title is also defined.
Fit…
Select the curve fitting routine to be applied to the data
(Point to Point, Linear Regression, Polynomial
Regression, Spline with smoothing factor or 4 Parameter
fitting).
Overlaying
Select several standard curves to display on one graph.
Interpolations
Calculate an X value vs. a known Y value. The
interpolated value can be displayed on the graphic and
within a table.
Recalculate
Recalculate the curve after standards have been edited
within the graphical data. Outlying points can be
removed from the curve by pointing and clicking the
mouse, then selecting Recalculate.
User's Guide to KC4
Using KC4: Step-by-Step
To use KC4, follow the steps shown below. Not all steps pertain to all
readers or to all user needs.
1. Install the security key (sometimes referred to as the 'hardlock')
on your computer.
2. Install KC4 on your computer.
3. Connect your microplate reader to your computer.
4. Start KC4.
5. Select System options (via the System Menu):
•
Set Reader options.
•
Set SuperUser options.
•
Set Reader Control options.
•
Perform reader Diagnostics.
6. Open an existing / Create a new Protocol (via the Protocol
menu):
•
Set Reading parameters.
•
Configure the plate layout to show the location of well
identifiers.
•
Specify curve fitting parameters.
•
Define Single and Multi Plate Transformations.
•
Specify Cutoffs and Validation criteria.
•
Specify Reporting and Exporting criteria.
7. Open an existing / Read a new plate (via the Data menu):
•
Enter plate descriptions.
•
Enter sample names.
•
Read the plate.
•
Print reports.
•
Export results data.
8. Start Biograph and analyze/manipulate the resulting curve.
Note: In routine applications, it is advised that standard or dilution curves
be inspected before further calculations are performed. Results can also be
validated using the Protocol|Validation option. Validation may indicate
whether the data (such as controls and standards) is valid or invalid.
User's Guide to KC4
Overview • 1-9
Technical Support
KC4 is backed by a superior support staff. If the software fails to function
perfectly, please contact the Technical Assistance Center (TAC).
Whichever method of contact you choose, please be prepared to provide the
following information:
•
The KC4 software version and revision numbers (such as
Version # 2.7, Rev # xx, found under the About menu).
•
The product serial number.
•
The specific steps which reproduce your problem.
•
Any error messages displayed on the screen.
•
A daytime phone number.
•
Your name and company information.
•
A fax number (if available).
Written Communication
You may write a letter with your comments and send it to:
BIO-TEK INSTRUMENTS, INC.
Technical Assistance Center
Highland Park, Box 998
Winooski, VT USA 05404-0998
BIO-TEK INSTRUMENTS, S.a.r.l.
B.P. 236
78052 St. Quentin Yvelines Cédex
France
Phone Support
You can telephone the Technical Assistance Center between 8:30 AM and
5:30 PM Eastern Standard Time (EST), and/or Customer Service between
8:00 AM and 5:30 PM EST, Monday through Friday, excluding holidays.
Bio-Tek Instruments Main Number
(802) 655-4040
Technical Assistance Center
(800) 242-4685
Customer Service
(888) 451-5171
1-10 • Overview
User's Guide to KC4
(Phone Support, continued)
International Customer Service & Service Department (in US)
(802) 655-4740
International Customer Service & Service Department (France)
(33) 1 39 30 2276
Facsimile Support
You may send a fax with your questions or requests for help 24 hours a day
to the following numbers:
Technical Assistance Center
(802) 655-3399
(33) 1 39 30 0987 (Europe)
Customer Service
(802) 655-7941
Electronic Communication
Electronic communication is available via:
E-MAIL
[email protected]
INTERNET Site
http://www.biotek.com
Bio-Tek on the Web
www.biotek.com
If you have an Internet browser installed on your computer, you can visit
Bio-Tek's World Wide Website, at www.biotek.com. Here you can find
information on hardware and software products, read application notes, and
more.
To visit the site from within KC4, select About|Bio-Tek on the Web, then
click on Bio-Tek Home Page. KC4 will automatically load your browser
and go directly to the site.
User's Guide to KC4
Overview • 1-11
1-12 • Overview
User's Guide to KC4
Installing the Software
Read Me First!
Complete the following to get KC4 up and running:
1. Ensure the computer on which KC4 will be installed meets the
minimum system requirements.
2. Install the KC4 security key.
3. Install KC4.
4. Connect your microplate reader to the computer.
5. Configure the reader and KC4 so that they can communicate
with each other.
Installing the Software covers steps 1 through 4. See Reader Selection in
the System Menu section for information on connecting a reader to the
computer and configuring the reader and KC4.
Remember to register your copy of KC4 with Bio-Tek to record your
warranty, and to receive information on upgrades and new products.
User's Guide to KC4
Installing the Software • 2-1
System Requirements
To run KC4, you will need the following:
•
Intel 486 processor / 33 MHz minimum (Pentium / 66 MHz or
greater is recommended)
•
Microsoft Windows 95 or Windows NT operating system
•
8 MB of RAM minimum (16 MB or more is recommended)
•
20 MB of available hard drive space for the KC4 program files
•
Additional available hard drive space for storing protocol and
plate data files
•
3 ½" high density diskette drive (1.44 MB) or CD ROM drive
•
Super VGA color monitor
•
Keyboard and mouse ports
•
Parallel port for installation of security key
•
Serial port if autoreader present
Installing the Security Key
Your KC4 software package includes a security “key”. This key permits
access to certain KC4 functions, including plate reading, printing, and data
storage. The security key is sometimes also referred to as the “hardlock”.
To install the KC4 security key, follow these steps in order:
1. If any other device (such as a printer cable) is attached to your
parallel port (LPT1), temporarily disconnect it.
2. Firmly press the male side of the security key connection into
the parallel port. Turn the device’s thumb screws gently using
either your fingers or a small screwdriver. Do not over-tighten.
3. With the security key in place, you can reattach the peripheral
device that was originally connected to this port by inserting its
cable into the back of the KC4 security key. Firmly tighten the
screws of the cable, if applicable, to ensure a good “seat.”
¾The security key allows for the transparent pass-through of
data from your computer to the peripheral device in most
cases.
2-2 • Installing the Software
User's Guide to KC4
Software Installation
To Install KC4:
1. Start Windows.
•
Note: If you are prompted with the Windows password
entry, enter a valid password. This helps to ensure a
complete installation. If you were not prompted with the
Windows password entry, disregard this note.
2. It is strongly recommended that you exit all Windows programs
before installing KC4.
3. Place Disk 1 in a 3.5" diskette drive or the CD in the CD ROM
drive.
4. Click the Windows Start button and select Run.
5. When the Run dialog box appears, type <drive>:setup.exe,
where <drive> represents the diskette drive.
•
Alternatively, use the Browse function to access the drive,
then click on setup.exe.
6. Click OK.
User's Guide to KC4
Installing the Software • 2-3
After a brief pause, the Welcome dialog will appear.
7. Click Next to continue. An informational dialog will appear.
8. Click Next to continue.
2-4 • Installing the Software
User's Guide to KC4
The Software License Agreement dialog will appear.
9. If you accept these terms, click Yes to continue. Otherwise,
click No to discontinue the software installation.
•
If you click Yes, the Choose Destination Location dialog
will appear.
10. The default location for the software is displayed. Click Next to
accept the default location, or click Browse to specify a
different location.
User's Guide to KC4
Installing the Software • 2-5
•
After a location has been specified, the Select Program
Folder dialog will appear.
11. Click Next to accept the default program folder name, or type in
a new one.
If you click Next, the software installation will begin. When the
installation is complete, the Setup Complete dialog will appear.
12. Click Finish. KC4 is now installed on your computer.
2-6 • Installing the Software
User's Guide to KC4
Connecting a Reader to the Computer
Before KC4 can be used to communicate with a microplate reader, the reader
must be properly connected to the computer on which KC4 is installed.
The following is a brief set of steps for connecting the reader to the
computer. For more detailed information, refer to the reader's user manual.
1. Attach the appropriate cable to an available serial port on the
back of the computer.
2. Plug the other end of the cable to the serial port on the reader.
3. Connect the power cable to the back or side of the reader.
4. Plug all power cords into grounded electrical outlets. Use of a
surge protector is strongly recommended.
5. Use KC4 to set up the communication parameters, as described
under Reader Selection in the System Menu section.
Starting KC4
1. From the Windows Start menu, access the Programs menu, then
the KC4 menu. Click on KC4.
To create a KC4 shortcut
icon for your Desktop:
¾ Open Windows
Explorer. Make the
window small so you can
see the Desktop in the
background.
¾ In the KC4 folder, click
and hold the right mouse
button on KC4_32.exe.
Drag the item onto the
Desktop and release the
mouse button.
¾ Select Create
Shortcut(s) Here.
User's Guide to KC4
Installing the Software • 2-7
2. The About KC4 informational dialog will appear.
Note: If the security key is not installed properly, this dialog
will show “Demo release” under the version number.
3. Click OK to continue.
Note: When you click OK, KC4 may attempt to communicate
with an attached microplate reader. If you have not yet attached
a reader, do not be concerned at this time if messages resembling
“NO READER RESPONSE” and “Error when getting
wavelength table from reader” appear. These messages will
disappear when a reader is properly connected and configured.
After you click OK, the KC4 main screen appears.
Review the next section, Getting Started to learn about KC4’s
menu system, sample files, and help system.
2-8 • Installing the Software
User's Guide to KC4
Getting Started
Main Screen
KC4's main screen contains the Title Bar, Menu Bar, Toolbar and Status
Bar.
Title Bar
The title bar shows the current open Data and Protocol files.
•
The first set of brackets [ ] embrace the Protocol file name
(extension PRT). An asterisk (*) indicates the protocol file has
been modified and not yet saved.
•
The second set of brackets [ ] embrace the Data file name
(extension PLA). An asterisk (*) indicates the data file has been
modified and not yet saved.
Menu Bar
The Menu Bar allows access to the following general categories:
User's Guide to KC4
•
The Data Menu contains plate data-specific functions relating to
KC4. These can be grouped into data storage and retrieval,
printing features, and data acquisition.
•
The Edit Menu provides specific editing Cut, Copy, and Paste
functions commonly found in Windows.
•
The Protocol Menu provides specific functions relating to
protocols. Functions can be grouped broadly into protocol
filing, protocol definitions and reporting.
Getting Started • 3-1
•
Display|Curve(s) starts the Biograph module if the protocol
defines curve-fitting parameters. Display|Validation displays
the results of the Validation conditions.
•
The features under the System Menu define global parameters
required for using KC4 and perform reader-specific functions.
Options include reader setup, SuperUser tasks, system setup, and
reader diagnostic utilities.
Toolbar
The Toolbar allows quick access to KC4 functions. Accessibility of a
particular Toolbar button depends on whether or not its function is
applicable to the current environment. For example, the Biograph button is
accessible only if results are displayed and a curve has been generated.
Initialize memory for a New Data file. Click this button before reading a
plate.
Read a plate (or enter data manually, or read data from a file). Before
clicking this button, ensure that the reader is connected to the computer, and
that the communication parameters are properly configured.
Save a data file. Data files have the extension *.PLA. Selection of the target
directory is possible using the Browse facility. Note: It is possible to save a
data file with its associated protocol as a *.GLB file. The protocol must be
in current memory at the time.
Print reports. Before clicking this button, click the Report button to design
the report format.
Enter Sample IDs associated with the plate. This can be done before a plate
is read, or after a plate read when results are displayed in KC4.
Define or modify reading parameters. If this button is inaccessible, select
Protocol|New Protocol to select a protocol type, then click Settings to
define the reading parameters.
Configure the plate layout by assigning a meaningful identifier to each well.
Typical identifiers include “BLK”, “STD”, and “SPL”. Standard
concentrations and dilutions are also defined here.
3-2 • Getting Started
User's Guide to KC4
Define or modify curve-fitting parameters for Quantitative, Curve
Comparison, Toxico, and Endotoxin protocols. Configurable parameters
include Data to Interpolate, Curve Fit Method, and Interpolation values.
Define or modify Single Plate Transformations. Typical uses include
Using specific wells as local blanks for other wells and calculating ratios
between wells. Note: To define or modify Multi Plate Transformations,
select Protocol|Advanced, then select Protocol|Multi Plate
Transformations.
Customize the report format. Reporting parameters defined here are stored
with the currently open protocol when it is saved.
Launch the Biograph application. This button is accessible only if results
are displayed and a curve has been successfully generated.
Control the attached reader's door/carrier movement or incubation. This
button is only accessible if these functions are supported by the current
reader.
Status Bar
The Status Bar displays the current KC4 status and any enabled keyboard
functions.
User's Guide to KC4
Getting Started • 3-3
Navigating the Help System
Most KC4 dialogs contain a Help button that, when clicked, opens an
informational Help dialog. In addition to this context-sensitive help, KC4
provides access to the entire Help system.
Select Help Index from the About menu to open the Help Topics dialog.
•
Double-click on a book
to see a topic's sub-topics.
•
Double-click on a page
to view the text for a topic.
•
Highlight a topic then click Open to view the text for the topic.
•
Click the Index tab to perform a keyword search.
•
Click the Find tab to search the entire help system for a specific
word or phrase.
•
Click Print to print the text for a topic.
¾Note: If printing a topic that contains an image, you may
need to adjust the Windows print properties for your printer.
If the image is distorted, try setting the Graphics Dithering
to None.
•
3-4 • Getting Started
Click Cancel to close the Help Topics dialog and return to KC4.
User's Guide to KC4
Sample Files
The KC4 installation program copies sample protocol and plate data files
into a “Samples” directory within the KC4 root directory. Reviewing sample
protocol and plate data files will help you to learn more about how to use
KC4.
User's Guide to KC4
•
Files with the extension *.PRT represent protocol files.
•
Files with the extension *.PLA represent plate data files.
•
Open protocol and plate data files with matching names, such as
TOXICO.PRT and TOXICO.PLA.
Getting Started • 3-5
3-6 • Getting Started
User's Guide to KC4
Data Menu
Data Menu Quick Reference
The following table provides a quick explanation of each option on the Data
menu.
Item
Description
Notes
New Plate
Create/define a new
plate.
If a plate is in memory before you
select this option, and it has not
already been saved, you will be
prompted to save it.
Open...
Open an existing plate
data file.
If a plate is in memory before you
select this option, and it has not
already been saved, you will be
prompted to save it.
Close
Close the current plate. You will be prompted to save
before the plate is closed.
Save
Save the current plate.
There are two file formats:
Data format. Data saved in the
file includes:
• Raw data sent by the reader
• Plate description (prompts,
comments, date/time of
reading)
• Sample names
• Reading parameters (reader
name, filter values, etc.).
• Files with this format have a
.PLA extension.
Data+Protocol format. With this
format, the protocol description is
also saved in the file. Files with
this format have a .GLB
extension.
User's Guide to KC4
Data Menu • 4-1
Save As
Save the current plate.
Save and supply the plate with a
new name or format (for example,
.PLA or .GLB)
Export
Export results.
If no export parameters have been
defined in the current protocol, a
dialog box appears, allowing you
to select data to be exported. You
then enter the pathname of the
exporting file.
Append to
Kinetic File...
Appends plate data
from one file to
another.
Allows plates to be read on a
discontinuous time basis, unlike
pure kinetic readings.
Print...
Print the report.
A window appears with the
option to print all or only a
portion of the report, by selecting
the pages to print, and other
parameters, as defined by
Protocol|Report.
Print Preview
Preview the report.
A Zoom In/Out feature is
available, as is the ability to view
one or two pages at a time.
Print Setup
Define the print setup
before printing.
Select the printer, paper size, and
paper orientation.
Plate Description... Set the description of
4-2 • Data Menu
This description includes:
the current plate.
• Reading date and time
• 6 customizable prompts. (Via
Protocol|Plate Description)
• 4 lines of comments. Press
Ctrl+Enter to insert a line-feed
in the comments.
Samples...
Define the names of
samples.
When the cursor is located on a
sample name that ends with a
figure (example: 'SMP01'), press
the down arrow key to
automatically increment the next
sample names ('SMP02', 'SMP03',
and so on).
Read Plate...
Read the current plate, If the plate has already been read,
enter data manually, or a message appears, confirming
read in data from a file. the re-read. If there is no protocol
loaded, a dialog box to define
reading parameters appears. Once
the reading parameters have been
defined, the plate description
dialog box displays.
Exit
Exit KC4.
Return to the Windows Program
Manager.
User's Guide to KC4
Working with Plates
This section describes accessing, generating, modifying, and saving plate
data files.
New Plate
Select Data|New Plate to clear memory to prepare for a new plate. If an
existing, but unsaved, plate is open, you are prompted to name and save the
data plate.
Open
Select Data|Open to open an existing stored plate data file.
•
Use the Look In drop-down list to search through folders.
•
Highlight a file and see its name in the File Name field.
•
To change the type of files being viewed, choose a different
option from the Files of Type drop-down list. Options include
KC4 Data (*.PLA), KC4 Data+Protocol (*.GLB), and All Files
(*.*).
•
Highlight the desired file, then click Open.
¾The retrieved data file will be displayed on a full screen
matrix.
¾Data reduction will be performed if a protocol is open.
User's Guide to KC4
Data Menu • 4-3
Close
Clicking
does not close
a plate data file.
Select Data|Close to close the current plate data file. If the file has not
already been named, the Save As dialog will appear requesting entry of a
file name.
Save
Select Data|Save to save the current open plate data file. If the file had been
saved previously, it is automatically saved using the existing name.
Save As
Select Data|Save As to save the current data file under a new name.
The default file name is
PLATE.PLA.
•
Select an existing file or enter a new file name.
•
If an existing file name is selected, KC4 will provide a warning
to avoid an accidental overwrite.
The existing saved file will
be overwritten if replaced.
¾Click Yes to continue with the overwrite.
¾Click No to rename the file before saving.
4-4 • Data Menu
User's Guide to KC4
Exporting Plate Data
KC4 can export plate data into a file that can then be used by any other
software program with the ability to read an ASCII text file. The text
delimiter used during the export can be selected under System|Setup.
Export parameters can be predefined within a protocol, or selected after a
plate has been read.
To export plate data:
•
Select System|Setup to select the Import/Export Delimiter.
Options include semicolon, comma, tab, or a custom character.
•
Select Data|Export.
•
If export parameters are not predefined in the current Protocol
then the following dialog appears:
The items under Available
Data can be selected and
added to the Exported
Data window. Like items
can be grouped together.
The text delimiter is
selected under
System|Setup.
¾See Protocol Menu, Export for information on selecting
export parameters.
¾See Appendix A for information on Export capabilities
when using KC4 in conjunction with a robotic system.
•
If the Export parameters are predefined in the protocol, selecting
Data|Export produces the Export As dialog.
¾Use this dialog to select or create the export file.
User's Guide to KC4
Data Menu • 4-5
Export File
Note: The exported data can be imported back into KC4. Before doing this,
the [ProtocolDescr], [ReadingParameter], [DataDescription], and other
informational sections must be removed from the text file, leaving only the
delimited raw data.
Sample Export File
[ProtocolDescr]
Name=D:\KC4\SAMPLES\SAMPLE PROTOCOL.PRT
Modified=No
[ReadingParameters]
PlateSize=6,8
PlateType=48 WELL PLATE
Wells=A1-F8
ReadBlankPlate=No
PathlengthCorrection=No
ReadBioCell=No
Filter1=405
Calibrate=No
[DataDescription]
Name=D:\KC4\SAMPLES\SAMPLE PLATE.PLA
ReadingDate=10/16/98 17:51:14
ReadingType=Manual
Temperature=
PromptTitles=Prompt #1:,Prompt #2:,Prompt #3:,Prompt #4:,Prompt
#5:,Prompt #6:
Prompts=,,,,,
Comment=
ReportTime=10/16/98 17:57:37
[Plate: M 405]
,1,2,3,4,5,6,7,8
A,0.005,0.004,0.009,0.010,0.479,0.485,0.016,0.014
B,0.010,0.013,0.236,0.238,0.509,0.511,0.014,0.020
C,0.156,0.152,0.412,0.415,0.392,0.389,0.487,0.492
D,0.278,0.276,0.478,0.469,0.224,0.220,0.510,0.505
E,0.359,0.362,0.567,0.568,0.482,0.479,0.008,0.010
F,0.481,0.479,0.359,0.362,0.101,0.099,0.410,0.415
[Plate: M 405 Corr.]
Blank=4.500E-3
,1,2,3,4,5,6,7,8
A,0.000,-0.001,0.004,0.005,0.474,0.480,0.012,0.009
B,0.005,0.008,0.231,0.233,0.505,0.507,0.009,0.016
C,0.152,0.147,0.407,0.410,0.388,0.385,0.482,0.487
D,0.274,0.272,0.473,0.464,0.220,0.216,0.506,0.501
E,0.354,0.357,0.563,0.564,0.477,0.474,0.003,0.005
F,0.476,0.474,0.354,0.357,0.097,0.095,0.405,0.410
[Plate: Concentrations]
,1,2,3,4,5,6,7,8
A,<8.0000,<8.0000,8.3472,8.4346,49.449,49.974,8.9593,8.7844
B,8.4346,8.6970,28.199,28.373,>52.000,>52.000,8.7844,9.3091
C,21.202,20.853,43.590,43.852,41.841,41.578,50.149,50.586
D,31.871,31.697,49.362,48.575,27.149,26.799,>52.000,51.723
E,38.955,39.217,>52.000,>52.000,49.711,49.449,8.2597,8.4346
F,49.624,49.449,38.955,39.217,16.393,16.218,43.415,43.852
[Validation: VALIDATION]
[Validation: Data type : M 405 Corr.]
,Condition,Status
1,STD1<STD2,VERIFIED
2,STD4<STD5,VERIFIED
4-6 • Data Menu
User's Guide to KC4
Append to Kinetic File
KC4 provides the option to create a kinetic file from individual endpoint
read plates. Each current plate can be appended to the previously read
plate. This type of file allows plates to be read on a discontinuous time base
unlike pure kinetic readings.
•
Alternatively, it is possible to append several kinetic readings to
one file. If a kinetic reading sequence is halted (ex. to inject
reagents) then it is possible to resume the kinetic reading and
append the resumed data onto an existing file.
•
The Append feature can be set to perform automatically after a
plate read. See Protocol Menu, Protocol Options for more
information.
To append data to a kinetic file:
•
After a plate is read, select Data|Append to Kinetic File.
•
The Append to Kinetic File dialog will appear:
¾The last opened plate file name will appear as the default.
Change the path and/or file name if desired.
¾Click New if this set of data represents the starting kinetic
read (i.e., if subsequent reads will be appended to this one).
If no file name has been entered, KC4 will produce the
standard Save As dialog.
¾Click Open to search through the directory structure to
locate the target file.
¾Click OK to continue. A dialog will appear requesting entry
of a Reading Time relative to the first reading.
The Append to Kinetic File
time format is defined
under System|Setup.
For a kinetic interval
greater than 99:59:59,
set the time format to
HHH:MM.
The default value represents the elapsed time since the last
plate read. Enter a new value if required.
User's Guide to KC4
Data Menu • 4-7
Printing Reports
After plate data has been acquired and calculations are complete, KC4 can
generate a user-customizable set of results reports. Reporting options are
selected and customized within each protocol by selecting
Protocol|Reports.
Print
Select Data|Print to print the reports selected in the protocol. The Print
dialog will appear.
•
To select a different printer, click the Printer Name drop-down
arrow.
•
To change print properties such as paper type and resolution,
click the Properties button.
•
To select a print range other than All, click Pages, then enter
the page range (e.g., from 2 to 4).
•
To print multiple copies of the reports, enter the desired number
of copies.
•
4-8 • Data Menu
Click OK to print the reports.
User's Guide to KC4
Print Preview
Select Data|Print Preview to see what the reports will look like before they
are printed. This facility ensures the format and layout are correct. The
layout may differ among printers.
User's Guide to KC4
•
Click Print to send the defined report to the default printer.
•
Click Next Page to view the next page in the report.
•
Click Prev Page to view the previous page in the report.
•
Click One Page / Two Page to toggle between viewing a single
page of the report or viewing two pages at a time.
•
Click Zoom In to get a closer look at a portion of the page.
•
Click Zoom Out to view the full page.
•
Click Close to close the preview and return to the results
display.
Data Menu • 4-9
Print Setup
To change the printer, paper size or source, or page orientation, select
Data|Print Setup to access a standard Windows Print Setup dialog.
4-10 • Data Menu
•
To select a different printer, click the Printer Name drop-down
arrow.
•
To change print properties such as paper type and resolution,
click the Properties button.
•
To select a different paper size, click the Paper Size drop-down
arrow.
•
To select a different paper source, click the Paper Source dropdown arrow.
•
To select the paper orientation, click the Portrait or Landscape
radio button.
•
Click OK to save the changes and return to the KC4 results
display.
User's Guide to KC4
Plate Description
The Plate Description dialog appears just prior to each plate read, to
provide the optional entry of plate-specific information.
Select Protocol|Plate
Description to customize
the data entry field names
that appear in this dialog.
•
Up to 6 free-text input fields can be customized via
Protocol|Plate Description.
User's Guide to KC4
•
The Reading Date and Time are automatically logged when the
plate is read.
•
This information is stored with the plate when Data|Save is
selected.
Data Menu • 4-11
Samples
Select Data|Samples to assign names to sample groups defined under
Protocol|Plate Layout.
Each sample ID can be entered manually, or the Auto Numbering feature
can be used to automatically increment ID suffix numbers.
•
The ID entered in cell #1 will be assigned to every replicate in
Sample group one, the ID in cell #2 will be assigned to every
replicate in Sample group two, and so on.
•
Up to 12 alphanumeric characters can be used for each ID.
Note: If a sample name cannot be printed in its entirety, it is
truncated and the character '\' is added at the beginning of the
name to indicate that the name has been truncated. For example:
'\LE001', '\LE002', etc.
•
To automatically number the sample IDs sequentially:
¾Check Auto Numbering.
¾In cell #1, type the identifying name and starting number
(e.g., Smp1) for Sample group #1. Leave the cursor in cell
#1.
¾Press the down arrow through as many cells as there are
sample groups on the plate. KC4 will automatically
increment the suffix number (Smp2, Smp3, and so on).
4-12 • Data Menu
User's Guide to KC4
•
Sample IDs can be entered using a barcode scanner attached to
the computer.
¾Place the cursor in cell #1 (or any empty cell).
¾Scan the item with the barcode scanner. The first 12
characters of the ID will appear in KC4.
¾Move the cursor down to the next empty cell, then scan the
next item.
Import Sample Names
The Import option allows you to import a sample name list from a text file.
Click Import, then locate and select the file to import.
•
The import file must contain one name label per line.
•
The number of IDs in the file should equal the number of
samples as defined under Protocol|Plate Layout.
If there are more IDs in the text file than samples defined on the
plate, the extra IDs at the end of the file will remain unused.
•
If a Sample ID in the text file exceeds 12 characters, only the
first 12 characters will be imported.
Export Sample Names
The Export option allows you to export the generated identification labels to
a text file for future use.
Enter the Sample IDs as required, then click Export to specify the export file
name.
•
The export file will contain one name label per line.
Printing Sample IDs
Click Print As Plate to print the Sample IDs in a matrix format
corresponding with the plate format selected in the protocol.
•
The plate must be configured via Protocol|Plate Layout prior to
using this feature.
Click Print As List to print the Sample IDs in a list format.
•
User's Guide to KC4
One Sample ID will be printed on each line.
Data Menu • 4-13
Read Plate
Select Data|Read Plate to initiate a plate read, or to enter data manually or
read data from a file.
If no protocol is open, a Reading parameters dialog appears. Parameters
available within this dialog are reader-specific.
If a protocol is open with
Reading parameters
defined, this dialog will not
appear.
•
Define the required Reading parameters, then click OK to
continue. The Plate Reading dialog will appear.
•
This dialog allows optional information and comments to be
added to the plate. This information will be filed along with the
data for future reference.
Select Protocol|Plate
Description to customize
the data entry field names
that appear in this dialog.
4-14 • Data Menu
User's Guide to KC4
¾Enter data as desired in the Plate Description fields.
¾Click Start Reading to initiate communication with the
attached microplate reader. Following a successful plate
read, the data will be transferred to KC4 and displayed on
screen.
¾Click Enter Manually to enter plate data using the keyboard.
See “Entering Plate Data Manually” for more information.
¾Click Read From File to read plate data from a stored file.
See “Reading Plate Data from a File” for more information.
Entering Plate Data Manually
KC4 provides the option to enter plate data manually, using the keyboard.
Typically this information comes from other instruments or from previously
acquired data.
Endpoint and kinetic data can be entered manually. With kinetic data, each
reading number set must be incremented to mimic the kinetic plate reading
cycle. The time base will be taken from the defined kinetic reading
parameters. Refer to Protocol|Reading|Kinetic Parameters for more
information.
Entering Endpoint Read Data
Use the up and down
arrow keys to move from
well to well in the matrix.
User's Guide to KC4
•
Place the cursor over the desired well and enter the OD (or
FU/LU if fluorescence/luminescence) value.
•
When pressing the Down Arrow, the cursor automatically jumps
from the end of one column to the top of the next (e.g., from H1
to A2).
•
Click OK to end the editing process and calculate results.
Data Menu • 4-15
•
For multiple wavelength reads, do the following to enter plate
data for all wavelengths:
¾Enter data for the first Data set (e.g., 405)
¾Click OK.
¾Select the next Data set (e.g., 550).
¾Click Change.
¾Enter the plate data for 550nm.
¾Repeat for the remaining wavelengths.
Entering Kinetic Read Data
Use the up and down
arrow keys to move from
well to well in the matrix.
4-16 • Data Menu
•
When this dialog first appears, data entry is expected for kinetic
Reading #1. Place the cursor over the desired well and enter the
OD (or FU/LU, if fluorescence/luminescence) value associated
with Reading #1.
•
When pressing the Down Arrow, the cursor automatically jumps
from the end of one column to the top of the next (e.g., from H1
to A2).
•
Click OK to end the editing process and calculate results.
•
To enter data for subsequent kinetic readings, update the
Reading # field, then click Change.
User's Guide to KC4
Reading Plate Data from a File
KC4 can read endpoint or kinetic data from an ASCII Text file. The file must
be formatted properly, and the data must be delimited with acceptable
separators.
To import Endpoint or Kinetic data:
•
Examine the text file to see what character is used to delimit the
data. Typical delimiters include semi-colon, comma, and tab.
•
Select System|Setup. Set the Import/Export Separator so that
it matches the character used within the text file.
•
If desired, create or open a protocol.
•
Select Data|Read Plate|Read from File, then select the desired
file. If the file is formatted properly, KC4 will read the data
then calculate results according to the protocol parameters.
Import File Format for an Endpoint Read
The file block size must correspond with the plate layout defined within the
protocol. For example, a 96-well plate has 8 rows and 12 columns, a 24-well
plate has 4 rows and 6 columns.
The following example represents the 96-well plate format:
0.003;1.409;0.483;2.022;1.463;1.200;0.876;2.240;2.057;1.867;0.435;2.147
1.776;1.284;0.760;0.037;0.229;0.911;0.368;0.415;2.471;1.114;0.298;0.012
0.022;0.945;1.329;1.428;1.504;1.518;0.416;1.658;1.127;0.880;0.143;1.519
1.958;2.007;1.300;0.755;2.190;1.817;2.390;2.314;1.348;0.356;1.155;0.588
2.156;0.524;1.949;2.109;2.492;2.499;1.529;0.981;0.666;0.743;2.100;0.059
0.940;0.232;1.693;0.141;0.022;2.297;0.690;0.682;1.470;1.728;2.094;1.816
1.212;0.513;1.859;1.171;1.145;2.373;1.861;0.271;1.498;0.963;1.838;1.522
1.431;0.903;0.379;0.563;1.063;2.007;1.293;2.475;1.879;0.864;0.422;1.643
Import File Format for a Kinetic Read
Note: Before importing a kinetic text file, create a protocol that specifies
the exact number of kinetic reads that will be imported.
The first line in the text file can optionally contain a time indicator. This
value will be stored in the protocol (overwriting protocol settings, if they are
different), and used for any kinetic plotting.
If the file contains no time indicators then the time parameters defined
within the Protocol|Reading parameters dialog will be used as defaults.
See the next page for a sample import file.
User's Guide to KC4
Data Menu • 4-17
The following example represents the kinetic data set generated from 5
readings, in the 24-well format:
To ensure a successful
import of all five reads
shown in this example,
create a protocol and
set the kinetic parameters
so that the Reading
Number is 5.
Time=00:00:00
0.000;0.007;0.013;0.020;0.026;0.033
0.004;0.011;0.017;0.024;0.030;0.037
0.008;0.014;0.021;0.028;0.034;0.041
0.012;0.018;0.025;0.032;0.038;0.045
Time=00:00:30
0.000;0.026;0.053;0.079;0.105;0.132
0.016;0.042;0.068;0.095;0.121;0.147
0.032;0.058;0.084;0.110;0.137;0.163
0.047;0.074;0.100;0.126;0.153;0.179
Time=00:01:00
0.000;0.059;0.118;0.178;0.237;0.296
0.036;0.095;0.154;0.213;0.272;0.331
0.071;0.130;0.189;0.249;0.308;0.367
0.107;0.166;0.225;0.284;0.343;0.402
Time=00:01:30
0.000;0.099;0.197;0.248;0.284;0.356
0.056;0.195;0.194;0.295;0.322;0.381
0.171;0.188;0.231;0.318;0.390;0.401
0.167;0.216;0.275;0.337;0.403;0.499
Time=00:02:00
0.000;0.176;0.251;0.299;0.378;0.446
0.117;0.281;0.295;0.375;0.397;0.441
0.257;0.275;0.331;0.388;0.438;0.482
0.239;0.296;0.369;0.402;0.492;0.557
4-18 • Data Menu
User's Guide to KC4
Edit Menu
Editing Tools
The Edit menu provides tools to copy virtually any set of data on the screen
in KC4 and paste it into a spreadsheet, word processor, database manager, or
text editor. Where appropriate, additional text editing functions are
available for cutting and pasting, in addition to copying. Under certain
circumstances, an Undo editing tool is available.
¾From within any data entry field or from within the results
dialogs, a click of the right mouse button will open an editing
menu.
Cut
Ctrl+X
Deletes selected data from the document and moves it to the clipboard.
This function is not always applicable.
Copy
Ctrl+C
Copies selected data to the clipboard. This function can be used with all
data displayed in a grid (microplate grid, statistics grid, etc.). The values
copied in the clipboard are separated by the character selected in the
System|Setup dialog.
Drag the mouse over the grid to select data, then select Edit|Copy, press
Ctrl+C, or click the right button of the mouse to select the Copy function.
Paste
Ctrl+V
User's Guide to KC4
Pastes data from the clipboard into the document. This function is not
always applicable.
Edit Menu • 5-1
Undo
Ctrl+Backspace
From any location within the Plate Layout dialog, click the right mouse
button and select Undo to erase the last change made to the configuration,
and restore any items that were modified by the last change.
If an edited formula within the Single or Multi Plate Transformation
dialog is highlighted, selecting Undo will cause the changed formula to
revert back to the original formula.
5-2 • Edit Menu
User's Guide to KC4
Protocol Menu
A Protocol contains instructions for controlling the attached microplate
reader and information required by KC4 to analyze data retrieved from the
reader (or for data entered manually or read from a file).
The following options are available under the Protocol menu:
To create a protocol, start
by selecting Protocol|New
Protocol.
History list shows the four
most recently used
protocols. Click on a
protocol name to open.
User's Guide to KC4
Protocol Menu • 6-1
Protocol Menu Quick Reference
The following table provides a quick explanation of each option on the
Protocol menu.
6-2 • Protocol Menu
Item
Description
Notes
New Protocol
Create a new protocol.
There are 5 categories of
protocols: Qualitative,
Quantitative, Curve
Comparison, Toxico, and
Endotoxins.
Open...
Open an existing protocol. KC4 provides a prompt to save
the current open protocol, if
applicable.
Close
Close the current protocol. KC4 provides a prompt to save
the open protocol.
Save
Save the current protocol.
--
Save As...
Save the current protocol
with a new name.
Use Save As to copy all of the
contents of an existing protocol
to a new protocol with a
different name.
Reading
Define reading
parameters.
Definable Reading parameters
depend on the currently selected
reader model.
Raw Data
Correction
Enable/Disable
corrections to raw data.
Options can include Blank Plate
Subtraction, Blank Wells
Subtraction, and Pathlength
Correction.
Kinetic Analysis
Select the primary (and
optional secondary) set of
data for kinetic analysis,
as well as Calculation
Type.
Available Calculation Types
include (“FU” indicates
Fluorescence Units, “LU”
indicates Luminescence Units):
•
•
•
•
Mean V
Max V
Mean Min/Max OD, FU, LU
Onset OD, FU, LU (basis
OD, FU, LU) / %
• Calculations based on userdefined formula
User's Guide to KC4
Spectrum Analysis
Select the set of data for
spectrum analysis.
Choose to calculate the
Min/Max ODs and the
wavelengths at Min/Max OD, or
create a special formula based
on the ODs at specified
wavelengths.
Plate Layout
Configure the plate layout
(position of standards,
controls, samples, etc.).
Available well identifiers are
dependent on the protocol type.
Single Plate
Transformations
Define and apply formulas Up to 3 levels of Single Plate
to one or more wells
Transformations can be defined.
within the plate. The
number of unique
formulas is limited only
by the number of wells on
the plate.
Multi-Plate
Transformation
Define calculations
Up to 3 levels of Multi Plate
between different sets of
Transformations can be defined.
plate data. Data-In
options include (but are
not limited to) raw data,
corrected data, and results
of Single Plate
Transformations.
Curves
Select the method to use
to calculate the standard
curve.
Report
Customize reports specific Design the report, read a plate,
to the protocol.
then select Data|Print Preview
to see the layout before printing.
Export
Select data to be exported
to a text file.
Optionally, select Data|Export
after a plate has been read.
Options
Select from several
automated functions.
Automated functions include:
Toggles between basic
and advanced protocol
functions.
Advanced protocol functions
include:
Advanced
Note: If you deselect the
Advanced mode after
defining advanced
parameters, KC4 warns
that the advanced settings
will be deleted.
User's Guide to KC4
Definable parameters include
Curve Origin, Curve Axes,
Curve Fit, and Interpolation.
•
•
•
•
•
•
•
•
•
Saving plate data
Appending to a kinetic file
Printing reports
Exporting plate data
Plate Description
Raw Data Correction
Multi Plate Transformations
Validation
Cutoffs
Protocol Menu • 6-3
Working With Protocols
This section provides information related to creating, changing, saving, and
accessing protocols.
New Protocol
Select Protocol|New Protocol to select the type of protocol to create:
KC4 automatically names
a newly created protocol
NONAME.PRT.
•
Qualitative: Collect absorbance, fluorescence, or luminescence
data. Use controls to define cutoffs for determining the status of
samples.
•
Quantitative: Create standard curves, and use known standards
to assign concentrations to unknown samples.
•
Curve Comparison: Create sample dilution curves and
interpolate response values.
•
Toxico: Use dilution curves to interpolate percentage response
values.
•
Endotoxin: Use known samples and spiked samples to calculate
endotoxin concentrations.
Open Protocol
Select Protocol|Open to open an existing protocol:
6-4 • Protocol Menu
•
To open a protocol, highlight the desired file then click Open.
•
Use the icons at the top of the dialog to search for the desired
protocol.
•
Protocol files have a *.PRT extension.
User's Guide to KC4
Modifying a Protocol
To modify an existing protocol, begin by opening the desired protocol.
When a protocol is
modified, an asterisk
appears next to its name
as a reminder to save
the protocol.
Once the protocol is open, use the Protocol pull-down menu to select a
category. Protocol categories are listed in a logical order of priority. For
example, the Plate Layout parameters should be defined before
Transformations are created and applied.
Several Toolbar buttons correspond with protocol categories:
Click Settings to access the Reading dialog, to define
parameters such as reading method, wavelengths, plate
type, and incubation. Note: Select the correct reader
via System|Readers before defining reading
parameters.
Click Layout to assign well identifiers and define
standard concentrations. Note: Select the plate
format (if applicable) in the Reading dialog before
assigning well identifiers.
Click Curves to define curve fitting parameters.
Ensure that any pre-reading parameters, blank wells,
and raw data correction parameters are defined before
selecting the curve parameter Data to Interpolate.
Click Transf. to access the Single Plate
Transformations dialog. The Multi Plate
Transformations dialog must be accessed from the
Protocol menu. Ensure that any pre-reading
parameters, blank wells, and raw data correction
parameters are defined before defining and applying
transformations.
Click Report to customize reports specifically for this
protocol. All protocol parameters should be defined
before selecting report options.
Note: Select Protocol|Advanced to access the advanced protocol
functions: Plate Description, Raw Data Correction, Multi Plate
Transformations, Validation, and Cutoffs.
¾Warning! If a protocol is defined with advanced protocol
functions, deselecting Advanced produces a warning message:
User's Guide to KC4
Protocol Menu • 6-5
Close Protocol
Select Protocol|Close to close an open protocol. If the protocol has been
modified or is unnamed, KC4 will prompt you to save the file:
•
Click Yes to save the new or modified information and close the
protocol.
•
Click No to close the protocol without saving changes.
•
Click Cancel to close this dialog and return to the open protocol.
The new or modified information is retained but not yet saved.
Save Protocol / Save As...
Select Protocol|Save or Protocol|Save As to save the open protocol.
will store the protocol under its current name or will ask for a name if
one does not yet exist. Save As can be used to copy all of the contents of an
existing protocol to a new protocol with a different name.
Save
6-6 • Protocol Menu
•
Use the icons at the top of the dialog to access the desired folder
in which to store the protocol.
•
Highlight an existing protocol or enter a new name in the File
Name field.
•
Click Save to save the protocol, or click cancel to close this
dialog without saving.
User's Guide to KC4
Plate Description
Just prior to a plate read, KC4 automatically presents a dialog containing up
to six free-text fields for the optional entry of information (such as
'Technician Name' or 'Lot Number') to be stored with the plate data.
Select Protocol|Plate Description dialog to customize the prompts for these
free-text fields. Note: Plate Description is an “advanced” protocol option.
Select Protocol|Advanced if Plate Description is not initally visible in the
Protocol menu.
Text entered here
substitutes the Prompt #
text seen in the Data|Plate
Description or Data|Read
Plate screen.
•
To customize a prompt, simply overwrite the default text in the
Prompt # field.
•
Up to 15 alphanumeric characters can be entered in each field.
¾If the Plate Description fields are defined as shown above,
the following dialog appears when Data|Read Plate is
selected:
User's Guide to KC4
Protocol Menu • 6-7
Defining Reading Parameters
Select Protocol|Reading to define plate-reading parameters. The
parameters available within the Reading dialog are specific to the reader
selected under System|Readers.
The parameters available
within this dialog are
reader-specific.
This example shows the
Reading dialog for the
ELx808-I reader.
The complete set of Reading parameters provided by KC4 are described
below and on the following pages.
Reading Type
The available Reading Type options can include Endpoint, Kinetic, and
Spectrum (reader-dependent).
6-8 • Protocol Menu
•
During an Endpoint Read, every well on the plate is read once
with one (single), two (dual), or multiple wavelengths.
•
During a Kinetic Read, every well on the plate is read a defined
number of times with one, two, or multiple wavelengths. Click
the Kinetic button to define specific kinetic parameters.
•
During a Spectrum Read, multiple readings are taken across a
wavelength range. The objective is to plot a graph to display
absorbance or transmission vs. wavelength.
User's Guide to KC4
Reader Model
The name of the currently selected reader model and the communication port
used are displayed under Reader. The reader and communication
parameters are defined via the System menu.
•
If the current reader is the FL600, three choices are presented for
method of reading: Absorbance, Fluorescence, and
Luminescence.
Kinetic Parameters
Clicking the Kinetic button opens the Kinetic dialog.
•
Run Time: Defines the total duration of the kinetic run. The
maximum duration is 48 hours.
•
Reading Number: Represents the total number of readings in
the kinetic scan. The maximum number of kinetic reads is 300.
•
Interval: Defines the time interval between individual plate
reads during the kinetic run.
Minimum kinetic intervals vary due to several factors, including
reader type and protocol parameters such as the number of
wavelengths and plate strips/wells specified, the plate format,
whether or not shaking is selected, and so on. If you enter a
kinetic interval that is too low for the reader type and for the
protocol parameters defined, KC4 provides a message stating the
minimum interval that can be entered.
For certain reader models, when you click OK to close the
Kinetic dialog, KC4 will attempt to communicate with the
reader to “ask” what the minimum kinetic interval should be. If
the reader is not connected at that time, another “request” will be
made just prior to the plate read, and you may be required to
modify the protocol.
Reading modes are set via
the reader's front panel.
Refer to the reader's user
manual for more
information.
User's Guide to KC4
For all readers except the PowerWave, PowerWavex, and
µQuant, the minimum kinetic intervals are based on reading in
Normal mode. When KC4 communicates with these readers to
request the minimum kinetic interval, the returned value is based
on the current reading mode, which can be Normal or Rapid.
Protocol Menu • 6-9
Scales for Kinetic Reads
By default with the Kinetic Reading Type, the Scales parameters
automatically calculate the best box size for the kinetic reactions.
Kinetic scales parameters are defined within the Kinetic parameters dialog.
•
The Scales parameter defaults are set as follows:
min t is set to a zero time value
max t is set to the Run Time value
min y is set to -1.0000 OD (or 0 FU)
max y is set to 5.0000 OD (or 99999 FU)
¾The default values can be manually overridden by
deselecting the Auto checkboxes.
•
Individual Well Auto Scaling individually adjusts the min/max
t and min/max y settings for each well to best display each
well's curve. It overrides the settings of the min/max t and
min/max y.
¾Note: This only affects the scale of an individual zoomed
well. The kinetic curves display, showing all wells on the
plate, is not affected.
Kinetic Parameters (FL600)
The Kinetic dialog contains some special parameters for the FL600.
Note: To adjust the time format for Kinetics, select System|Setup.
•
In Plate Mode, the FL600 repeatedly reads a whole or partial
plate for a specified number of times and for up to 6 filter sets.
•
In Well Mode, the FL600 repeatedly reads a well for a specified
number of times for up to 2 filter sets.
¾Well Mode is typically used for fast kinetic reactions.
6-10 • Protocol Menu
User's Guide to KC4
¾Check Eject between well to eject the assay plate between
well readings. This feature is useful, for example, for
accommodating reagent addition.
The configuration of
Reading parameters such
as number of Filter Sets,
Plate Type, and Shaking
options can affect the
minimum kinetic interval.
•
Run Time defines the duration of the total kinetic read.
•
Interval defines the time interval for repeat reading of the plate,
section of wells, or individual well. When the OK button is
selected to close the dialog box, KC4 automatically checks the
validity of the Interval value.
•
KC4 automatically calculates the Reading Number by dividing
the Run Time by the Interval.
•
By default, the Scales feature automatically calculates the best
box size for the kinetic reactions.
¾For the absorbance mode, min t is automatically set to a zero
time value, min y is set to -1.0000 OD, max t is set to the
Run Time value, and max y is set to 5.0000 OD.
¾For the fluorescence and luminescence modes, min t and
min y are automatically set to zero values, max t is set to the
Run Time value, and max y is set to 99999 FU/LU.
¾The default values can be manually overridden by
deselecting the Auto checkboxes.
•
Individual Well Auto Scaling individually adjusts the "min/max
t" and "min/max y" settings for each well to best display each
well's curve. It overrides the settings of the "min/max t" and
"min/max y".
Filters/Wavelengths
The reader-dependent Filters/Wavelengths parameters can include:
•
Meas. Filter: Select the measurement filter for a single- or dualwavelength read.
•
Refr. Filter: Select the reference filter for a dual-wavelength
read.
•
Wavelengths: For readers that support up to six wavelengths
per read, select from one to six unique wavelengths a single- or
multi-wavelength read, or select Start, Stop, and Step values for
a spectrum read. See Wavelengths for more information.
•
User's Guide to KC4
Filter Sets (FL600): Select up to six filter sets for absorbance,
fluorescence, or luminescence readings. See Filter Sets
(FL600).
Protocol Menu • 6-11
Wavelengths
For readers that support multiple wavelength reads, the Wavelengths set of
parameters is available within the Reading parameters dialog.
For Endpoint and Kinetic reads:
•
Click the radio button associated with the desired number of
wavelengths.
•
to open a list of available wavelengths, then click on a
Click
wavelength to select it.
¾If the desired wavelength is missing from the list, type the
value directly into the wavelength field.
•
(Optional) If the reader is connected, clicking Calibrate will
update KC4’s wavelength table and the reader’s internal
wavelength table.
¾If KC4’s wavelength table already matches the reader’s
wavelength table, calibration is not necessary.
¾If calibration is necessary but the reader is not connected
right now, calibration will perform automatically when a
plate read begins.
For readers that support spectral scanning, the Wavelengths set of
parameters changes when a Spectrum read is selected.
For Spectrum reads:
•
Define the wavelength range by entering the Start and Stop
values, in nanometers.
•
Define the reading interval by entering the Step value, in
nanometers.
¾For example, if the Start, Stop, and Step values are 300,
600, and 50 nm, respectively, the plate will be read at 300,
350, 400, 450, 500, 550, and 600 nm.
•
6-12 • Protocol Menu
(Optional) Select Calibrate:
User's Guide to KC4
¾If Calibrate is selected, the reader will always perform
calibration at the wavelengths specified in the protocol, just
prior to plate reading.
¾If Calibrate is not selected, the reader will calibrate at only
those wavelengths specified in the protocol that have not yet
been calibrated since the reader was turned on.
Scales for Spectrum Reads
By default with the Spectrum Reading Type, the Scales feature
automatically calculates the best box size for the spectrum curves.
Click the Scales button to open the Scales dialog:
•
The Scales parameter defaults are set as follows:
min w is set to the Start Wavelength
max w is set to the Stop Wavelength
min y is set to -1.0000 OD
max y is set to 5.0000 OD
¾The default values can be manually overridden by
deselecting the Auto checkboxes.
•
Individual Well Auto Scaling individually adjusts the min/max
w and min/max y settings for each well to best display each
well's curve. It overrides the settings of the min/max w and
min/max y.
Plate Options
Some reader models offer various plate options that allow for the selection
of a particular plate type, and/or to define the exact strips or wells to be
read.
Plate Type
For readers that support multiple plate types, the correct type must be
selected to ensure accurate results.
User's Guide to KC4
Protocol Menu • 6-13
•
To select a plate type, click on
available plate types.
to choose from a list of
¾For the FL600, the list of available plate types correlates
with the list of plate formats maintained under System|Plate
Formats.
Strip Selection
Certain readers support reading a subset of the entire plate, based on vertical,
contiguous microplate strips.
To specify the range of strips to be read:
•
Enter the starting strip number in the First Strip field.
•
Enter the ending strip number in the Last Strip field.
Well Selection
Certain readers support reading a subset of the entire plate, based on
contiguous microplate wells.
To specify the range of wells to be read:
•
If applicable, select the appropriate plate type. The default Last
Well field value will automatically update to match the selected
plate type.
•
Set the First Well and Last Well fields to indicate the range of
wells to be read.
¾If a plate is only partly filled with strips, change the First
and Last Well values to specify that a portion of the plate be
read. For example, if First Well is set to A1 and Last Well is
set to B3, KC4 will read a “rectangle” of wells: A1, A2, A3,
B1, B2, and B3.
•
Some readers also support a Reading Direction setting, to
choose between a horizontal plate read (A1-A12, B1-B12, …
H1-H12), or a vertical plate read (A1-H1, A2-H2, … A12-H12).
¾The faster method for reading a full plate is horizontally.
¾The faster method for reading an 8 well strip is vertically.
6-14 • Protocol Menu
User's Guide to KC4
Plate Shaking
If plate shaking is available with the currently selected reader, the Shaking
parameters are accessible.
•
For Endpoint and Spectrum readings, shaking occurs
immediately before the plate is read.
•
For Kinetic readings, shaking can occur before the first reading
only, or before every reading.
•
Intensity: Acceptable values include:
0
1
2
3
4
•
No Shaking
Low
Medium
High
Variable (not supported by all readers that support shaking)
Duration: Defines the absolute time period for shaking, in
seconds.
•
Before First / Before Every Reading: Defines when the
shaking will occur.
¾For non-kinetic reads, shaking occurs before the first
reading.
¾For kinetic reads, shaking can occur before the first read or
before every read.
User's Guide to KC4
Protocol Menu • 6-15
Temperature Control
For readers that support computer-controlled incubation, the Temperature
Control parameters are accessible:
The Lag Time feature is
available for all reader
models, regardless of
incubation capabilities.
•
Select Yes to indicate plate incubation.
•
Select No to indicate no incubation (default).
•
Check Pre-Heating to pre-heat the incubator before the plate
carrier is drawn in.
•
Enter the desired set point Temperature in °C.
•
Define the Lag Time to elapse before the reader initiates the first
reading cycle.
¾On most readers, the plate carrier will enter the reading
chamber and wait for the Lag Time to elapse, then the plate
will be read.
¾The valid entry range for most readers is 00:00:00 to
00:59:59. For the FL600, the maximum Lag Time is
08:00:00.
Pre-Readings
Depending on the selected Reader model and Reading Type, one or more
Pre-Reading options may be available.
Pre-Read Blank Plate
Check Pre-Read Blank Plate to perform two reads at each defined
wavelength. The first read will be performed on an empty (or “blank”) plate,
and the second on the actual microplate. Note: The “Blank Plate” can
represent a dry empty plate or a plate filled with solution for blanking.
Note: KC4 automatically
subtracts the Blank Plate
data from the actual
microplate data unless set
otherwise.
See Raw Data Correction
for more information.
6-16 • Protocol Menu
•
For Endpoint and Kinetic reads, the Blank Plate is read at each
wavelength defined in the protocol.
•
For Spectrum reads, the Blank Plate is scanned using the
wavelength range defined in the protocol.
•
The Blank Plate data is subtracted from the actual microplate
data if the Blank Plate Subtraction option is selected in the
Raw Data Corrections dialog (advanced protocol option).
User's Guide to KC4
Pathlength Correction
Once a plate has been
read, the Pre-Reading
options cannot be
deselected. You can,
however, choose to disable
the Pathlength Correction
calculations (as well as
Blank Plate or Blank Wells
Subtraction) via the
Protocol|Raw Data
Corrections dialog.
Check Pathlength Correction to normalize absorbances to 1 cm. This
option is only available for readers that support multiple wavelength reads
up to 999 nm.
If Pathlength Correction is enabled, KC4 first calculates the pathlength for
each well. The absorbance in each well is then divided by the pathlength,
resulting in the corrected absorbance.
KC4 automatically reports the “Corrected” data after Pathlength Correction
is applied. A Multi Plate Transformation can be easily created to report the
pathlength for each well. See tbd for more information.
•
Each well's pathlength is calculated as follows:
(A977 nm - A900 nm)well
(A977 nm - A900 nm)1.0 cm water
¾The default Test and Reference Wavelengths of 977 and
900 nm, respectively, can be changed by clicking Options
and defining different wavelength values.
¾KC4 uses a default value of 0.18 for the absorbance of water
at 1 cm (977 nm - 900 nm). This value can be changed by
clicking Options and defining a different Absorbance of
Solution at 1 cm.
Alternatively, click Bio-Cell to calculate the absorbance of
water at 1 cm value using Bio-Tek's Bio-Cell plate. Use
the drop-down menu to select the location of the cuvette in
the plate.
¾KC4 automatically reports the Corrected Data after
pathlength correction is applied. To report the Pathlength
for each well, create a simple Multi Plate Transformation.
See tbd for more information.
•
The absorbance in each well is then corrected according to the
following formula:
(Awavelength)well
pathlength of well
¾If a Blank Plate is pre-read, and/or if blank subtraction is
performed, select the advanced protocol option Raw Data
Correction to control the data used in this formula.
User's Guide to KC4
Protocol Menu • 6-17
•
Click Options to alter the Test or Reference Wavelength values,
or to change the pathlength constant.
¾The valid range for the Test and Reference Wavelengths
fields is 200-999 nm.
¾The default Absorbance of solution at 1 cm value is 0.18.
To use a different constant value when calculating
pathlengths, simply enter the desired value.
Extended Range
If supported by the reader, use the Extended Range feature to extend the
reporting of optical density values retrieved from the microplate reader.
6-18 • Protocol Menu
•
If Extended Range is disabled, KC4 will collect optical density
values up to 3.000 OD, and report values greater than 3.000 OD
as '?????'.
•
If Extended Range is enabled, KC4 will report OD values
greater than 3.000 OD.
User's Guide to KC4
Filter Sets (FL600)
The FL600 can perform absorbance, fluorescence, or luminescence reads
with up to six uniquely defined Filter Sets.
There are many applications for the use of multiple filter sets, including:
•
Assays that have more than one fluorophore in the well.
•
Comparison of results between the top and bottom optics
positions.
•
Comparison of results between rapid and static mode.
•
Comparison of results with varying static mode settings.
•
Comparison of results with varying sensitivity settings.
To define Filter Sets:
1. Ensure the protocol is appropriately set for Absorbance,
Fluorescence, or Luminescence mode.
2. Select the Filter Set radio button corresponding with the number
of sets required.
3. Define the wavelengths:
Absorbance mode:
¾For each filter set, use the drop-down lists to select the
desired wavelength.
Fluorescence mode:
¾For each filter set, use the drop-down lists to select the
Excitation and Emission wavelengths.
¾Note: The Emission wavelength is typically longer than the
Excitation wavelength. To prevent damage to the reader,
KC4 prohibits the saving of a protocol with overlapping
Excitation and Emission wavelengths, or provides a warning
if the wavelengths are considered to be too close, but does
not prohibit the protocol from being saved.
User's Guide to KC4
Protocol Menu • 6-19
Luminescence mode:
¾For each filter set, use the drop-down lists to select the
Emission wavelength.
¾Select Lum/E to indicate an empty position in the filter
wheel.
4. Specify the Optics Position for each individual filter set
(Endpoint Readings), or for all filter sets (Kinetic Readings).
The FL600 is equipped with two optical probes, one above
(Top) and one below (Bottom) the assay plate. This
accommodates the top reading of homogeneous solutions in
solid black plates and bottom reading of cellular assays in clear
bottomed plates.
Tips:
¾Evaluate the reading position for each filter set by selecting
two (or more) filter sets, one each for the Top and Bottom
optics positions.
¾The bottom position is advantageous for homogeneous
solutions exciting at 470 nm or above, and if the assay plate
bottom does not exhibit high background fluorescence.
¾The preferred Optics Position for Absorbance readings is
Bottom. KC4 will provide a warning if Top is selected,
although the selection will not be prohibited.
¾The preferred Optics Position for Luminescence readings is
Top.
5. Fluorescence or Luminescence mode only: Specify the
Sensitivity setting for each individual filter set. Valid settings
are 0 and integers from 25 to 255. 'Auto' indicates that the Auto
Sensitivity Adjustment feature has been enabled.
The signal from the range of fluorescence assays can range from
very strong to very weak. The sensitivity of the photomultiplier
(PMT) can be adjusted for a particular assay to ensure that the
signals from all wells fall within the appropriate dynamic range
of 0 to 99,999 fluorescence or luminescence units (F.U. or
L.U.).
Tips:
¾Typical assays using 96- (or fewer) well plates require
sensitivities between 75 and 190. Assays using 384-well or
other higher-density plates will likely require much higher
sensitivities due to narrower optical probes.
¾To determine the optimum sensitivity for an assay, define
multiple filter sets with the same parameters, varying only
the sensitivity setting.
6-20 • Protocol Menu
User's Guide to KC4
¾As the sensitivity setting increases, so will the fluorescence
values. If many wells result in “OVRFLW” (indicating F.U.
or L.U. values greater than 99,999), the sensitivity setting is
probably too high.
¾If each well contains more than one fluorophore, one of
which might give brighter or weaker results, assign each
filter set its own sensitivity.
¾If the field shows 'Auto', the Automatic Sensitivity
Adjustment feature is enabled. For more information, refer
to the next section, Measurement Options.
6. Click the Options… button to specify the Measurement
Options for each individual filter set (Endpoint Readings), or
once for all filter sets (Kinetic Readings). See below.
Measurement Options (FL600)
The selected Measurement Mode instructs the FL600 to read the assay plate
rapidly, or to slow down the reading and provide measurements of increasing
precision and hence sensitivity.
To define Measurement Options for a particular filter set (Endpoint) or for
all filter sets (Kinetic), click the Options button associated with desired
filter set.
A Static Mode reading
performs more slowly than
a Rapid Mode reading, but
the CV's are usually better.
•
Rapid Mode reads each well while the assay plate is in motion.
This provides the fastest reading.
¾The FL600 is equipped with fluorescent plastic compounds
called reference wells which help provide the best results
when using Rapid Mode to read the assay plate.
See General Options (FL600) for more information on
reading the reference wells with Rapid Mode.
User's Guide to KC4
Protocol Menu • 6-21
For Absorbance reading,
the selection of Static
Mode with 10 samples per
well and a delay before
sampling of 0.35 is highly
recommended.
•
Static Mode performs a specified number of reads in each well,
with a specified delay between each read. Nb Samples, Delay
before sampling, and Delay between samples pertain
specifically to Static Mode.
¾Nb Samples is the number of measurements taken per well.
Valid options are 1, 2, 3, 4, 10, 20, 50, and 100
measurements, and the value reported for each well
represents the average of its measurements.
Usually, the more samples per well the better the CV’s.
Selecting more than 10 samples per well, however, may
result in only marginal improvement. 10 therefore is the
recommended combination of sensitivity and speed.
¾Delay before sampling is the pause time of the optical
probe before the reading is taken. Valid options are 0.10,
0.35, 0.50, and 2.00 seconds.
0.35 seconds is the recommended setting to ensure the probe
is definitely stopped for measurement and for the minimum
amount of time.
¾Delay between samples is the time delay between samples.
The valid range is 0 to 2550 milliseconds. Longer delays
between samples may result in better CV’s between
replicates.
Automatic Sensitivity Adjustment
Enable Automatic Sensitivity Adjustment to let KC4 automatically select
the most appropriate sensitivity value for a particular filter set.
If Automatic Sensitivity Adjustment is enabled in a protocol, KC4 instructs
the FL600 to perform several pre-readings on the plate to determine the
optimal sensitivity. The “optimal” sensitivity is based on the best signal-tonoise ratio obtained during these pre-readings.
To assist KC4 with sensitivity selection, certain wells on the plate must be
designated as Low, Experimental, and High Wells, and limit-setting
fluorescence/luminescence Base and High Values must be specified:
6-22 • Protocol Menu
User's Guide to KC4
1. Designate certain wells on the plate as Low, Experimental, and
High Wells.
•
Enter a single well location (ex. A1) or a “rectangle”
(ex. B1-C2) of well locations.
•
Low Wells are usually blank wells, buffer wells, or negative
controls, and are expected to yield a low F.U. or L.U.
•
Experimental Wells are usually low standards or low
positive controls, and are expected to yield a mid-range F.U.
or L.U.
•
High Wells are usually the highest standards or high positive
controls, and are expected to yield a high (but not overrange)
F.U. or L.U.
2. Specify the Base and High Values.
•
The Base Value is the fluorescence or luminescence value
that the Low and Experimental Wells must exceed during
sensitivity determination. This ensures that the plate
produces values that are large enough to be meaningful.
•
The High Value is the fluorescence or luminescence value
that the High Well(s) should approach but not exceed.
The default setting is 90,000 F.U./L.U, and the maximum
value is 99,999. Practically, this value could be anywhere
from 10,000 to 90,000.
General Options (FL600)
KC4 provides options for controlling certain reader functions preceding and
during plate reads, such as lamp control and plate ejection.
Click the Options button from within the Reading Parameters dialog to open
the General Options dialog. The available/recommended settings vary,
depending on the reading mode.
User's Guide to KC4
Protocol Menu • 6-23
Absorbance mode:
•
Enable Eject between Filter Set Read to:
¾Perform an initial reading of a blank plate, followed by the
assay plate(s) at some later time.
¾Provide the ability to add reagents to the same plate between
readings.
¾Enable reading up to 6 different plates with one protocol and
plate file.
¾This feature is available for Endpoint reads only.
•
Skip reference wells is selected for absorbance readings, and
cannot be changed.
•
Enable Check Lamp before measurement to check lamp
output prior to measurement. This feature is selected by default.
Deselecting this feature speeds up the reading process, however,
a lamp output failure may not be detected.
•
Enable Set Lamp Off during measurement to allow for the
reading of an unexcited signal. This feature is deselected by
default.
Fluorescence mode:
•
Enable Eject between Filter Set Read to:
¾Perform an initial reading of a blank plate, followed by the
assay plate(s) at some later time.
¾Provide the ability to add reagents to the same plate between
readings.
¾Enable reading up to 6 different plates with one protocol and
plate file.
¾This feature is available for Endpoint reads only.
•
Skip reference wells is deselected by default.
The FL600 is equipped with fluorescent plastic compounds call
reference wells which help provide the best results when using
Rapid Mode to read the assay plate. Skip reference wells should
be deselected for use with Rapid Mode reads.
See Reading the Reference Wells on the next page for more
information.
6-24 • Protocol Menu
•
Enable Check Lamp before measurement to check lamp
output prior to measurement. This feature is selected by default.
Deselecting this feature speeds up the reading process, however,
a lamp output failure may not be detected.
•
Enable Set Lamp Off during measurement to allow for the
reading of an unexcited signal. This feature is deselected by
default.
User's Guide to KC4
Luminescence mode:
•
Enable Eject between Filter Set Read to:
¾Perform an initial reading of a blank plate, followed by the
assay plate(s) at some later time.
¾Provide the ability to add reagents to the same plate between
readings.
¾Enable reading up to 6 different plates with one protocol and
plate file.
¾This feature is available for Endpoint reads only.
•
Skip reference wells is deselected for luminescence readings,
and cannot be changed.
•
Check Lamp before measurement is deselected by default.
The lamp does not need to be checked before measurement.
•
Set Lamp Off during measurement is selected for
luminescence readings, and cannot be changed. The lamp is
turned off during measurement to avoid damage to the
photomultiplier.
Reading the Reference Wells
It is strongly recommended that you read the reference wells if Rapid Mode
is enabled. If, however, reading the Reference Wells must be skipped,
consider the following:
•
During an Endpoint read, when prompted to “Insert plate and
press OK”, do so within 60 seconds.
•
If a Lag Time is specified for an Endpoint or Kinetic read, the
maximum recommended time is 60 seconds. If a longer lag time
is required, read the reference wells first or use Static Mode to
read the plate.
•
During a Kinetic read, the maximum time interval is 60 seconds.
If a longer time interval is required, read the reference wells first
or use Static Mode to read the plate.
If the Reference Wells are skipped and the above time limits are exceeded,
the FL600 can provide unexpectedly high fluorescence values in the first
well that is read on the assay plate. Usually the first well is A1 but since the
software provides the ability to change the starting well, the first well can be
anywhere on the plate.
User's Guide to KC4
Protocol Menu • 6-25
Scanning (FL600)
Reading multiple points across a single microwell (scanning) more
accurately assesses cellular assays than simply reading once in the center of
the well. Scanning is available for Endpoint reads only.
Following a Scanning plate read, KC4 reports:
•
The raw data for each reading point.
•
An average of the raw data in each well.
•
A graphical display of the scanned wells (shown above).
To enable scanning, click the Scanning button within the Reading
Parameters dialog.
Note: If the Scanning button is dimmed and inaccessible, well scanning
cannot be performed for the currently defined reading parameters. See Well
Scanning Determination on the next page for more information.
6-26 • Protocol Menu
User's Guide to KC4
•
The Read Matrix Size represents the number of scans to be
performed on each well. Valid entries are odd integers from
1 to 25.
The FL600 scans across a well in 2 dimensions. The maximum
number of scans depends on the well diameter and probe size.
The valid entries are odd to ensure that the center point is always
read.
•
Individual Well Color Auto Scaling determines how the color
palette is built when the reading points are graphically displayed
(Picture Mode). Either the whole plate or each individual well is
taken into account when determining the fluorescence values
associated with the darkest shade and the lightest shade.
If the plate will yield values ranging from very low to very high,
enable Individual Well Color Auto Scaling to help with the
visual differentiation between points within each well.
Well Scanning Determination
KC4 automatically evaluates the current combination of Reading parameters
to determine whether or not well scanning can be performed:
•
KC4 calculates the maximum number of scans that can be
performed on one well. This calculation is based on the probe
size defined in System|Readers|Configuration, and the well
diameter of the currently specified Plate Type.
Important! Do not change the Probe Size value in
System|Readers|Configuration unless the actual probe in the
reader is changed.
•
The maximum number of scans per well equals:
well diameter / (probe diameter/2) - 1
rounded down to the nearest odd integer.
•
If the Scanning button is dimmed and inaccessible, well
scanning is prohibited for the current probe size / plate type
combination.
For example:
¾If the reader is equipped with 5 mm probes and the current
plate type is 96 WELL PLATE, well scanning cannot be
performed.
¾If the reader is equipped with 5 mm probes and the current
plate is Nunclon 24 flat bottom, well scanning can be
performed, and the maximum number of scans per well is 5.
User's Guide to KC4
Protocol Menu • 6-27
Raw Data Correction
“Raw Data” – the optical density or fluorescence unit values retrieved from a
microplate reader – can be corrected using Blank Plate Subtraction, Blank
Wells Subtraction, and/or Pathlength Correction. The advanced protocol
option Raw Data Correction controls the use of these correction options.
•
Each Raw Data Correction option is accessible if it applies to
the configured protocol:
¾Blank Plate Subtraction is accessible if “Pre-Read Blank
Plate” is selected in the Reading parameters dialog.
¾Blank Wells Subtraction is accessible if one or more blank
wells are defined under Plate Layout.
¾Pathlength Correction is accessible if it is selected in the
Reading parameters dialog.
•
By default, each accessible Raw Data Correction option is
enabled.
See “Pre-Readings” on
page 6-16 for more
information on Pathlength
Correction.
•
Raw Data Correction calculations are performed in the order
Blank Plate Subtraction, Blank Wells Subtraction, then
Pathlength Correction.
•
The following example describes the plate processing and
calculation steps performed when a protocol specifies Pre-Read
Blank Plate, Pathlength Correction with Bio-Cell, and the plate
is configured with blank wells. This example is for explanation
only – the typical application does not use all three raw data
correction options.
Processing steps:
1. The Blank Plate is read at every wavelength defined in the
Reading parameters dialog.
2. The Bio-Cell is read at 977 nm and 900 nm.
3. The Sample Plate is read at 977 nm and 900 nm.
4. The Sample Plate is read at every wavelength defined in the
Reading parameters dialog.
6-28 • Protocol Menu
User's Guide to KC4
Calculation steps performed for each wavelength defined in the
Reading parameters dialog:
1. The basis of calculations begins with the raw Sample Plate
readings.
2. The Blank Plate readings are subtracted from the Sample
Plate readings. The result of this calculation becomes the
basis for step 3.
3. The mean of the blank wells is calculated and subtracted
from every well on the plate. The result of this calculation
becomes the basis for step 4.
4. The value in each well is multiplied by the difference in
readings of the Bio-Cell at 977 nm and 900 nm. The result
of this calculation becomes the basis for step 5.
5. The value in each well is divided by the difference in
readings of the Sample Plate at 977 nm and 900 nm. The
result of this calculation is the final corrected value.
•
The image below shows the data sets available following a
three-wavelength read with Blank Plate Subtraction, Blank
Wells Subtraction, and Pathlength Correction enabled:
¾M 977 (SP) and M 900 (SP) contain the values of the Sample
Plate read at 977 nm and 900 nm, respectively (necessary for
pathlength correction calculation).
¾Measurement numbers (M#1, M#2, M#3) correspond with
the wavelength selection order in the Reading parameters
dialog.
¾M#1 405, M#2 450, and M#3 630 contain the raw Sample
Plate values at 405 nm, 450 nm, and 630 nm.
User's Guide to KC4
Protocol Menu • 6-29
¾M#1 405 Blank, M#2 450 Blank, and M#3 630 Blank
contain the values calculated by subtracting the Blank Plate
readings at the specified wavelength from the raw Sample
Plate readings at the specified wavelength. These values do
not represent Blank Well Subtraction.
¾M#1 405 Corr., M#2 450 Corr., and M#3 630 Corr. contain
the final values after all Raw Data Correction options have
been applied.
Blank Plate Subtraction
The “blank plate” can
represent an empty dry
plate or a plate filled with
solution for blanking.
With most readers, the “Pre-Read Blank Plate” option is available within the
Reading parameters dialog. If this option is selected, a blank plate is read
before the sample plate, at the wavelengths defined in the protocol. KC4
then calculates the difference in measurements between the two sets of data.
If Blank Plate Subtraction is enabled:
•
Endpoint read: For each wavelength defined in the protocol,
KC4 subtracts the value of each well of the blank plate from the
corresponding well of the sample plate.
•
Kinetic read: For each wavelength defined in the protocol,
within each kinetic read, KC4 subtracts the value of each well of
the blank plate from the corresponding wells of the sample plate.
•
Spectrum read: At each wavelength read within the defined
spectrum range, KC4 subtracts the value of each well of the
blank plate from the corresponding well and wavelength of the
sample plate.
Blank Wells Subtraction
Within any protocol, if one or more wells are defined as 'Blank' under Plate
Layout, the Blank Wells Subtraction Raw Data Correction parameter
automatically becomes enabled.
If Blank Wells Subtraction is enabled:
•
Endpoint read: For each wavelength defined in the protocol,
the average of the blank well(s) is subtracted from every well on
the plate.
Note: For readers that do not accept multiple (> 2) wavelengths,
if a dual-wavelength read is defined, KC4 automatically
calculates the Delta OD first. The average of the Delta OD
values in the blank wells is then subtracted from every well on
the plate.
•
6-30 • Protocol Menu
Kinetic read: For each wavelength defined in the protocol,
within each kinetic read, the average of the blank wells is
subtracted from every well on the plate.
User's Guide to KC4
Note: For readers that do not accept multiple (> 2) wavelengths,
if a dual-wavelength read is defined, KC4 automatically
calculates the Delta OD first. Within each kinetic read, the
average of the Delta OD values in the blank wells is then
subtracted from every well on the plate.
•
Spectrum read: A blank average is calculated for each
wavelength in the spectrum reading range. The blank average
for each wavelength is then subtracted from the absorbance read
at the corresponding wavelength in each well.
Pathlength Correction
With readers that support multiple-wavelength reads up to 999 nm, the
“Pathlength Correction” option is available within the Reading parameters
dialog. If this option is selected, absorbances are normalized to 1 cm.
If Pathlength Correction is enabled, KC4 first calculates the pathlength for
each well. The absorbance in each well is then divided by the pathlength,
resulting in the corrected absorbance.
See Pre-Readings under Defining Reading Parameters for more
information.
User's Guide to KC4
Protocol Menu • 6-31
Kinetic Analysis
Use Kinetic Analysis to select pre-data reduction parameters for kinetic
reads.
This option becomes available within the Protocol menu when the Kinetic
read method is selected.
Data to Process
This option allows the selection of the type of data to be analyzed, i.e., the
measured response value for kinetic analysis. The pull-down list displays
the available data types.
Typical use of this feature:
When two data sets are
selected, the kinetic curves
are automatically overlaid
in the kinetic graphs and in
the kinetic zooms.
6-32 • Protocol Menu
•
To blank data prior to the kinetic slope calculation. First, define
one or more blank wells in the Plate Layout, and make sure
Blank Wells Subtraction is enabled in the Raw Data Correction
dialog. Then, select '<wavelength> Corr.' from the Kinetic
Analysis Data to Process pull-down list.
•
With some readers it is possible to perform kinetic reading with
multiple filters. In this case, two levels of Data to Process
options can be selected (e.g., '405' and '550') to perform and
compare kinetic calculations.
User's Guide to KC4
Mean V
Mean V is the calculated value of the mean slope. KC4 determines the
mean slope by calculating a linear regression on points that are in the
calculation zone. To set the calculation zone, select Mean V, then click on
Calculation Options.
Calculation Zone
Typically, the Calculation Zone is adjusted to ignore erroneous data point(s)
generated at the beginning of a kinetic assay due to assay “noise”.
•
From t / To t : Defines a calculation zone based on time. The
default values span the defined kinetic Run Time.
•
From # / To # : Defines a calculation zone based on number of
reads. The default values include all reading points.
•
mOD or OD dependent on kinetic dynamic range.
•
Per sec or Per sec dependent on rate order.
•
Per min or Per min dependent on rate order.
•
Per hour or Per hour dependent on rate order.
Units
2
2
2
Max V
With Max V, the calculated value is the maximum slope. KC4 calculates the
maximum slope as follows:
User's Guide to KC4
•
Starting at the first point in the calculation zone, KC4 evaluates
n points and calculates the slope among these n points.
•
Next, KC4 repeats the operation, starting at the second point in
the calculation zone, and repeats again, starting at the third
point, and so on.
•
KC4 then compares all calculated slopes to determine the
maximum slope.
•
KC4 will register the Delta t time in the middle of the point
where the V max is calculated.
Protocol Menu • 6-33
To set the calculation zone, select Max V, then click Calculation Options.
Calculation Zone
Typically, the Calculation Zone is adjusted to ignore erroneous data point(s)
generated at the beginning of a kinetic assay due to assay “noise”.
•
From t / To t : Defines a calculation zone based on time. The
default values span the defined kinetic Run Time.
•
From # / To # : Defines a calculation zone based on number of
reads. The default values include all reading points.
V calculation on n points
•
This option defines the number of successive points to be
included in the calculation of the maximum slope.
•
The minimum number of points is 2. The maximum number of
points is the total number of points within the calculation zone.
•
Only the points defined within the calculation zone are used.
•
mOD or OD dependent on kinetic dynamic range.
•
Per sec or Per sec dependent on rate order.
•
Per min or Per min dependent on rate order.
•
Per hour or Per hour dependent on rate order.
Units
2
2
2
Mean Min/Max
The Mean Min OD is the mean minimum OD based on n points. The Mean
Max OD is the mean maximum OD based on n points. KC4 calculates the
Mean Min and Mean Max ODs as follows:
•
6-34 • Protocol Menu
Starting at the first point in the calculation zone, KC4 evaluates
n points and calculates the mean among these n points.
User's Guide to KC4
•
Next, KC4 repeats the operation, starting at the second point in
the calculation zone, and repeats again, starting at the third
point, and so on.
•
KC4 then compares all of the calculated means to determine the
minimum and maximum of these values.
•
KC4 also calculates the Delta t at Min/Max OD.
•
Note: For fluorescence and luminescence reads, KC4 performs
calculations based on fluorescence units (Mean Min/Max F.U.)
and luminescence units (Mean Min/Max L.U.), respectively.
To set the calculation zone, select Mean Min/Max OD, then click
Calculation Options.
Calculation Zone
Typically, the Calculation Zone is adjusted to ignore erroneous data point(s)
generated at the beginning of a kinetic assay due to assay “noise”.
•
From t / To t : Defines a calculation zone based on time. The
default values span the defined kinetic Run Time.
•
From # / To # : Defines a calculation zone based on number of
reads. The default values include all reading points.
Mean Min/Max on n points
•
Selects the number of points to be included in the average
calculation.
Onset OD / Onset OD %
The calculated value is the time when the OD reaches a set decrease/increase
(Onset OD), that can be defined by an absolute value or a relative value (%),
compared with a reference OD (the Basis Value). You can either manually
enter this value, or have KC4 calculate it (mean of the n first points). By
default n=1, thus the Basis Value is the OD of the first point.
•
KC4 returns the time for each well to pass the Onset OD value.
•
Note: For fluorescence and luminescence reads, KC4 performs
calculations based on fluorescence units (Onset F.U.) and
luminescence units (Onset L.U.), respectively.
User's Guide to KC4
Protocol Menu • 6-35
To set the calculation zone, select Onset OD or Onset OD %, then click
Calculation Options.
Basis Value Calculation
•
Mean of the n first points: For relative determination of the
Basis Value.
•
OD = : For absolute determination of the Basis Value.
Examples:
Onset OD=0.5, Basis Value=Mean of first 2 points:
KC4 calculates the time that is required for the OD to increase
by 0.5 OD, compared with the mean of the 2 first points of the
kinetics.
Onset OD=15%, Basis Value=Mean of the first point:
KC4 calculates the time that is required for the OD to increase
by 15%, compared with the first point of the kinetics.
Onset OD=1.6, Basis Value=0:
KC4 calculates the time that is required for the OD to reach the
value of 1.6 (Basis Value + Onset OD).
Formula
Select Formula to define a formula to calculate a value using data from
individual reading points from the same well. Designate an individual
reading point using the identifier Rn, where n represents the reading number,
then enter a Result Name in which to store the formula results. This name
can be used as a Data-In setting in Transformations. Formula examples:
•
The formula R10-R5 calculates the difference in readings
between data points #10 and #5.
•
The formula R# (e.g., R1) extracts a single reading from each
well for endpoint data reduction.
No Calculation
This option, if selected, overrides any calculations on the kinetic data, and
allows the display of kinetic graphics without data reduction.
•
6-36 • Protocol Menu
No Calculation is typically used to display overlaid curves.
User's Guide to KC4
Spectrum Analysis
KC4 provides the option for performing additional calculations on spectral
data. This is available for Spectrum reading types only.
Open the Spectrum Analysis dialog by selecting Protocol|Spectrum
Analysis:
Data to Process
Select up to two levels of data to be used in calculations.
•
When two data sets are selected, the spectrum curves are
automatically overlaid in the spectrum graphs and in the
spectrum zooms.
Min/Max
Select Min/Max to determine the minimum and maximum values for the
points defined within the calculation zone. This is the default setting. Upon
completion of a read, the following is reported for each well:
•
Minimum OD and Wavelength at Minimum OD
•
Maximum OD and Wavelength at Maximum OD
If the Calculation Type is set for Min/Max, certain calculation parameters
may be customized. Access the Spectrum Analysis - Min/Max dialog by
clicking the Calculation Options button from within the
Protocol|Spectrum Analysis dialog:
User's Guide to KC4
Protocol Menu • 6-37
•
From w and To w allow the specification of a range of
wavelengths upon which to perform calculations. The default
From w and To w values correspond with the Start and Stop
values in the Protocol|Reading Parameters dialog.
•
From # and To # allow the specification of a range of read
numbers upon which to perform calculations. The default From
# and To # correspond with the total number of wavelengths to
be read, based on the Start, Stop, and Step values in the
Protocol|Reading Parameters dialog.
Example: Start/Stop/Step values set to 400/600/10,
respectively. The default From # and To # values are 1 and 21,
indicating a calculation zone from 400-600nm. Setting From #
to 2, and To # to 18 would change the calculation zone to 410570nm.
•
Mean Min/Max specifies the number of successive wavelength
readings to be averaged prior to the determination of the
Minimum and Maximum OD values.
Formula
Check the Formula radio button to define a formula to structure calculations
by a formula. Enter the formula into the long field.
Example: The formula W1+W2+W3 calculates the sum of the ODs for the
readings at wavelengths 1, 2, and 3. If the plate is read from 400 to 410nm
in 2nm increments, W1 will correspond with the OD at 400nm, W2 with the
OD at 402nm, and W3 with the OD at 404nm.
6-38 • Protocol Menu
•
Enter a Result Name in which to store the formula results. This
name can be used as a Data-In variable in Transformations.
•
Mean Min/Max specifies the number of successive wavelength
readings to be averaged prior to the determination of the
Minimum and Maximum OD values.
User's Guide to KC4
Plate Layout
Select Protocol|Plate Layout to design the plate layout by selecting and
specifying locations for standards, controls, blanks and samples, as well as
the number of replicates for each.
For readers that support
multiple plate formats,
ensure that the correct
format is selected in the
Reading parameters dialog
before configuring the
plate.
•
Select the required identifier from the pull-down menu.
•
Enter the number of replicates and replicate direction.
•
Choose the fill direction. Clicking Auto will automatically
increment the identifier in the selected wells.
•
Click and drag to select all the wells that you wish to have the
selected identifier.
Identifiers
Identifiers are fixed format labels that associate the value sent by the reader
with a particular defined function. Identifiers form the essential link
between data and result calculation. Correct assignment is essential to
establish meaningful expression of the results.
The following describes all identifiers supported by KC4:
User's Guide to KC4
SPLn
Sample, n denotes the incremental sample number.
BLK
A global Blank, which is automatically subtracted from all
wells.
BLKn
A local Blank, which must be subtracted using a Single-Plate
Transformation.
STDn
Standard, n denotes the incremental standard number.
Standards indicate the wells to be used for standard curve
generation.
Protocol Menu • 6-39
CTRLn
Control, n denotes the incremental control number.
NEGn
Negative control, n denotes the incremental control number.
POSn
Positive control, n denotes the incremental control number.
SPKn
Spike (Endotoxins), n denotes the known spike concentration
applied to the sample.
Empty
Empty well, typically selected and applied to delete an
incorrectly placed identifier.
The Protocol Type (defined when Protocol|New Protocol was selected),
determines which identifiers will be available for Plate Layout.
Protocol Specific Identifiers - Qualitative
Qualitative data reduction allows for the categorization of data into pre-
defined groups. Group thresholds are delimited using fixed or variable
values. Fixed thresholds or cut-offs are defined from validated standardized
calibrators while variable cut-offs are defined using factors calculated during
run-time to compensate for operational variations.
Qualitative protocols may utilize the following identifiers:
Sample:
SPL1 to SPL96
Specific Blank:
BLK1 to BLK96
General Blank:
BLK
Positive Control:
POS1 to POS12
Negative Control:
NEG1 to NEG12
Control:
CTRL1 to CTRL12
Protocol Specific Identifiers - Quantitative
Quantitative data reduction allows for unitary evaluation of data. Known
standard concentrations allow calculation of a single standard curve
generated from data using curve fitting methods. Sample data is
subsequently interpolated and reported in meaningful scientific
nomenclature.
Quantitative protocols may utilize the following identifiers:
6-40 • Protocol Menu
Standard:
STD1 to STD20
Sample:
SPL1 to SPL96
Local Blank:
BLK1 to BLK96
Global Blank:
BLK
Positive Control:
POS1 to POS12
Negative Control:
NEG1 to NEG12
Control:
CTRL1 to CTRL12
User's Guide to KC4
Protocol Specific Identifiers - Curve Comparison
Curve Comparison allows for 2 or more dilution curves to be compared
simultaneously, usually graphically on the same axes. Interpolation allows
the user to quantify the relative differences between nominated curves.
•
Each Sample must represent a dilution series of at least 2
dilutions. Ideally the minimum number of replicates must be 2
wells per dilution.
•
Curve Comparison ideally includes a local blank for each
sample dilution sequence represented on the plate. The local
blanks will represent the zero base lines used for the generation
of the individual curves.
•
Curve Comparison usually requires local controls representing
the maximum upper range of the dilution curve.
Curve Comparison protocols may utilize the following identifiers:
Sample:
SPL1 to SPL12 (sample curves to compare)
Local Blank:
BLK1 to BLK12
Global Blank:
BLK
Control:
CTRL1 to CTRL12
Protocol Specific Identifiers - Toxico
Toxico protocols allow the calculation of percentage (%) L.D. 50 from
graphically presented dilution curves. Controls define the upper dynamic
range as 100% response and the blanks define the lower dynamic range as
the 0% response. The transformation formulas are automatically defined
after the Plate Layout has been created, the response of the samples are
expressed as % response relative to the controls.
User's Guide to KC4
•
Each Sample must represent a dilution series of at least 2
dilutions. Ideally the minimum number of replicates must be 2
wells per dilution.
•
Toxico ideally must include a local blank for each sample
dilution sequence represented on the plate. The local blanks will
represent the zero percent base lines used for the generation of
the individual curves.
•
Toxico requires local controls with known dilutions for each
sample dilution sequence represented on the plate. The control
represents the 100% upper range of the dilution curve.
•
Blanks represent 0% toxicity. Controls represent 100% toxicity.
Protocol Menu • 6-41
Toxico protocols may utilize the following identifiers:
Sample:
SPL1 to SPL12 (products to study)
Local Blank:
BLK1 to BLK12 (blanks specific to a product)
Global Blank:
BLK
Local Control:
CTRL1 to CTRL12 (controls specific to a product)
Global Control:
CTRL
Protocol Specific Identifiers - Endotoxin
Endotoxin tests require the use of a standard curve to evaluate known
diluted samples. Each sample is usually accompanied by a spike.
•
Samples can have only one dilution. If you have several
dilutions, then consider them as several samples.
•
Spikes allow the calculation of the recovery %. After data
evaluation, the recovery % is displayed in the Statistics report.
It is defined as (Conc[Spike Well]-Conc[Sample Well])/Spike
Value.
Endotoxin protocols may utilize the following identifiers:
6-42 • Protocol Menu
Spike:
SPKn (n denotes spike concentration applied to the
sample)
Sample:
SPL1 to SPL96
Standard:
STD1 to STD12
Local Blank:
BLK1 to BLK96
Global Blank:
BLK
Positive Control:
POS1 to POS12
Negative Control:
NEG1 to NEG12
Local Control:
CTRL1 to CTRL12
User's Guide to KC4
Dilutions
KC4 provides the ability to assign dilutions to wells.
To define dilutions, click the Dilutions button at the bottom of the Plate
Layout dialog after defining Identifiers.
Dilution values can be
assigned to control and
sample wells in Curve
Comparison and Toxico
protocols, and to sample
wells in Quantitative and
Endotoxin protocols.
To assign dilutions and manually increase the dilution values:
•
Enter the starting dilution value in the Dilution field.
•
Specify the number of replicates in the Replicate field, then
select the direction (Horizontal or Vertical).
•
Click the wells requiring the defined dilution.
•
To assign the next dilution, change the value in the Dilution
field, then click one or more wells.
To assign dilutions and let KC4 increase the dilution values:
•
Enter the starting dilution in the Dilution field.
•
Specify the number of replicates in the Replicate field, then
select the direction (Horizontal or Vertical).
•
Check a Filling method of Auto Increm. or Auto Factor.
Choose Auto Increm. to increase each dilution exactly by a
defined value. Choose Auto Factor to increase dilutions by a
defined factor value.
User's Guide to KC4
•
Enter the Increment or Factor value, as appropriate.
•
Select the direction (Horizontal or Vertical) in which increasing
dilutions will be mapped.
•
Highlight a block of contiguous wells to assign dilutions.
Protocol Menu • 6-43
Standards
If the plate layout contains Standard wells, concentrations must be assigned.
To define concentrations, click the Standards button at the bottom of the
Plate Layout dialog after defining Identifiers.
Within the Standards dialog, the Standard identifiers defined for the plate
appear in sequential order in the Standards column. Concentrations are
entered in the Concentration column.
Concentrations can be entered manually, or assigned using the automatic
numbering feature:
6-44 • Protocol Menu
•
Choose the method by which to increase the concentration
values for subsequent standards: Auto Increment to increase
each standard exactly by a defined value, or Auto Factor to
increase standards by a defined factor value.
•
Enter the Increment or Factor value, as appropriate.
•
Place the cursor in the first cell in the Concentration column. If
the desired starting concentration is a number other than 0, enter
the number.
•
Using the down arrow key, move the cursor down the
Concentration column to assign concentrations.
•
If desired, enter the unit of measure in the Unit field.
User's Guide to KC4
Spikes
If the plate layout contains Spike wells, concentrations must be assigned.
To define concentrations, click the Spikes button at the bottom of the Plate
Layout dialog after defining Identifiers.
Within the Spikes dialog, the Spike identifiers defined for the plate appear
in sequential order in the Spikes column. Concentrations are entered in the
Concentration column.
Concentrations can be entered manually, or assigned using the automatic
numbering feature:
User's Guide to KC4
•
Choose the method by which to increase the concentration
values for subsequent spikes: Auto Increment to increase each
spike exactly by a defined value, or Auto Factor to increase
spikes by a defined factor value.
•
Enter the Increment or Factor value, as appropriate.
•
Place the cursor in the first cell in the Concentration column. If
the desired starting concentration is a number other than 0, enter
the number.
•
Using the down arrow key, move the cursor down the
Concentration column to assign concentrations.
•
If desired, enter the unit of measure in the Unit field.
Protocol Menu • 6-45
Conc./Dil.
In Curve Comparison and Toxico protocols, every curve is represented by a
sample (SPL1, SPL2 etc.). In order for a sample curve to be generated, each
sample must contain at least 2 different Concentrations or Dilutions.
To assign Concentrations or Dilutions, click the Conc./Dil. button at the
bottom of the Plate Layout dialog:
To assign concentrations or dilutions and manually increase the values:
•
Enter the starting value in the Conc./Dil. field.
•
Specify the number of replicates in the Replicate field, then
select the direction (Horizontal or Vertical).
•
Click the wells requiring the defined concentration or dilution.
•
To assign the next value, change the value in the Conc./Dil.
field, then click one or more wells.
To assign concentrations or dilutions and let KC4 increase the values:
6-46 • Protocol Menu
•
Enter the starting concentration/dilution in the Conc./Dil. field.
•
Specify the number of replicates in the Replicate field, then
select the direction (Horizontal or Vertical).
•
Choose Auto Increm. to increase each concentration or dilution
exactly by a defined value. Choose Auto Factor to increase by a
defined factor value, then enter the Increment or Factor value,
as appropriate.
•
Select the direction (Horizontal or Vertical) in which increasing
values will be mapped.
•
Highlight a block of wells to assign concentrations or dilutions.
User's Guide to KC4
Single Plate Transformations
KC4 provides for up to three levels of Single Plate Transformations. The
three different levels of transformations may be interdependent, or
completely independent of each other. Typical uses include:
•
Using specific wells as local blanks for other wells.
•
Calculating ratios between wells.
The Single Plate Transformation dialog functions similarly to a
spreadsheet. The data entry template resembles a microplate in row x
column format. Formulas are entered in “cells” representing the wells
within the microplate.
To define Single Plate Transformations:
•
For readers that support multiple plate formats, ensure the
correct format is selected under Protocol|Reading.
•
Ensure that identifiers have been assigned under Protocol|Plate
Layout.
•
Select Protocol|Single Plate Transformations.
Click
to
define Single Plate
Transformations.
¾Place the cursor in a well and enter the desired formula. The
formula is visible in the Current Formula box at the bottom
of the dialog.
¾The Transf. # field indicates the current transformation
level. Level 1 transformations can be passed for further
manipulation into Level 2 and Level 3 transforms for
successive manipulation.
¾Data In represents the data set upon which the formulas will
be applied. The Data In choices will vary depending on the
defined protocol parameters.
User's Guide to KC4
Protocol Menu • 6-47
¾For kinetic protocols, a Kinetic checkbox is provided. If
this box is checked, the Data-In options represent plate data,
such as “M#1 405” or “M#2 630”. If this box is not
checked, the Data-In options represent the results of kinetic
calculations, such as “Max V” and “Coeff Reg”.
¾The Data Out field assigns a name to the transformed data.
The transformed data can then be used for further
calculations, including Multi Plate Transformations.
¾Select Copy Current Formula to copy the current formula
into one or more wells. Clicking on the title row or column
auto-fills the wells selected with the current formula.
Clicking on the top left corner auto-fills the whole plate.
¾Select Edit Well to edit the formula in a particular well.
¾Select Diff. Between Lines to perform subtraction between
rows, then select A-B or B-A.
¾Select Diff. Between Cols to perform subtraction between
columns, then select 1-2 or 2-1.
¾Click Init. All Transf. to reinitialize all Single Plate
Transformations to their most recently saved settings.
to expand the width of the columns, to view and
¾Click
access long formulas. The maximum expansion shows a
single column of wells in full screen.
¾Click
to contract the width of the columns.
Formula Syntax
The following tables contain symbols and functions that can be used in
Single-Plate Transformations:
Type of Symbol
Description/Example
Algebraic symbols
+,-, /,*,()
Figures (scientific notation)
2.45E-08
x or X
Represents the current well value.
coordinates of the well
Represents the value of a particular well,
such as A2 or H12.
Functions Applied
to the Identifier
MEAN[POS1]
6-48 • Protocol Menu
Description/Example
Represents the mean of identifier POS1.
'POS1' by itself also represents the mean
of all POS1 wells on the plate.
User's Guide to KC4
SD[NEG1]
Represents the standard deviation of all
valid wells in group NEG1.
CV[BLK]
Represents the coefficient of variation of all
valid wells in group BLK.
Advanced Mathematical
Functions
Description/Example
LOG(x)
Represents the log10 function.
POW(x;y)
Raises x to the power of y.
POW(10;x)
Calculates the Anti-Log of the current well.
SQRT(x)
Square root of x.
MIN(x1;x2;x3...)
Minimum value of defined variables.
Example: MIN(A1;B1;C1).
MAX(x1;x2;x3...)
Maximum value of defined variables.
Example: MAX(CV[CTRL1];CV[CTRL2]).
Formula Examples
CTRL1-3*SD[CTRL1]
The result of this formula is the mean of the wells containing the identifier
CTRL1 minus three Standard Deviations of these wells. It is the equivalent
of MEAN[CTRL1]-3*SD[CTRL1].
X+(A5+A6)/2
The result of this formula is the sum of the current well value and the mean
of the A5 and A6 wells.
LOG(X/MEAN[POS1])
The result of this formula will log the ratio of the current well over the mean
of POS1.
MAX(A1;A2;A3)-MIN(B1;B2;B3)
This formula calculates the difference between the maximum value of A1,
A2, A3 and minimum value of B1, B2, B3.
% = (SPLn-BLKn) / (CTRLn-BLKn)
For Toxicology protocols, the formulas that calculate toxicity percentages
are automatically generated, although they can be modified.
User's Guide to KC4
Protocol Menu • 6-49
Multi Plate Transformations
KC4 provides for up to three levels of Multi Plate Transformations. The
three different levels of transformations may be interdependent, or
completely independent of each other. In addition, the results of any Single
Plate Transformations can be incorporated into a Multi Plate
Transformation.
To define Multi Plate Transformations:
•
Ensure that the Reading Parameters, Kinetic Parameters,
Plate Layout, and Single Plate Transformations are defined as
required.
•
Select Protocol|Multi Plate Transformations.
¾The Transf. # field indicates the current transformation
level. Level 1 transformations can be passed for further
manipulation into Level 2 and Level 3 transforms for
successive manipulation.
¾Data In represents the data set upon which the formulas will
be applied. The Data In choices will vary depending on the
protocol type and reading parameters.
¾Within one Multi Plate Transformation, you can select up to
three discrete assay data sets for combined calculations.
You must select and position each data set in the appropriate
location holder (i.e., X1, X2 and X3). The data sets must be
of similar type.
¾You can select previously stored data files (these files must
be correctly formatted text files and only contain end-point
readings).
6-50 • Protocol Menu
User's Guide to KC4
¾For kinetic protocols, Reading # can be set to All, or from 1
to the total number of readings.
¾Use the location variables X1, X2 and X3 to construct the
Formula.
¾The Name field assigns a name to the transformed data and
saves the settings and formula. The transformed data can
then be used as the Data-In for further calculations,
including Kinetic Analysis and other Multi Plate
Transformations.
Syntax
The following table contains symbols that can be used in Multi Plate
Transformations:
Type of Symbol
Description/Example
Algebraic symbols
+,-, /,*,()
Figures (scientific notation)
2.45E-08
Automatic Formula Generation
For readers with multiple wavelength (> 2) reading capability, a default
Multi Plate Transformation is automatically created when exactly 2
wavelengths or filter sets are specified. These default parameters can be
changed.
•
The Protocol menu switches to Advanced Mode.
•
The Multi Plate Transformation dialog contains:
¾X1 = <Meas. OD#1>
¾X2 = <Meas. OD#2>
¾Formula = X1 - X2
¾Data Out = Delta OD
•
In addition, if the protocol is Kinetic:
¾The 'Kinetic' checkbox becomes enabled.
¾The Reading # fields for both X1 and X2 are set to All.
Note: Once the Reading Parameters dialog is saved and closed with 2
filter sets/wavelengths specified, the Delta OD Multi-Plate Transformation
will not automatically 'clear' if the protocol is then changed to 1 or 3-6 filter
sets/wavelengths. The user must either deselect the Advanced option to
'clear' the Multi-Plate Transformation, or open the Multi Plate
Transformation dialog and manually empty/modify the fields.
User's Guide to KC4
Protocol Menu • 6-51
Curves
Select Protocol|Curves to defined curve fitting parameters. Curve
parameters can also be modified while a plate data file is open.
Curve Description
Select Protocol|Curves to select the data to be used for curve fit generation,
and to select the origin of the curve.
Refer to the Biograph
module for information on
storing curves.
•
The Data to Interpolate setting represents the type of data to be
used for curve fitting.
•
Select Current Plate if the standard curve will be generated
using data from the current plate.
•
Select File if the standard curve will be based on a previously
stored curve. The retrieved curve is used for quantitative data
generated from the current plate.
¾Click Browse... to search for the desired file. The file must
have a *.GPH extension.
6-52 • Protocol Menu
User's Guide to KC4
Curve Axes
By default, the x- and y-Axis data is transformed linearly, and the
Extrapolation Factor is set to 1.1. These options can be modified by
selecting Protocol|Curves|Axes.
•
Select either Lin or Log for the transformation of x axis or y
axis data.
•
If Display Grid is checked, the graph will be displayed and
printed with gridlines.
•
The text in the x Axis and y Axis Title fields appears on
standard curve displays and reports.
¾To modify a title (e.g., “% toxicity” or “Delta OD”), simply
overwrite the existing text.
Inappropriate use of the
Extrapolation Factor
feature may invalidate
results generated from
data falling into the
extrapolation zone.
User's Guide to KC4
•
The Extrapolation Factor enables you to extend the abscissa
range of the curve fitting routine beyond the limits of the
standard data points generated from the assay. See Defining
Extrapolation for more information.
Care must be exercised in the use of extrapolation as the implied
assumption is that the relationship of the x and y variables is
valid outside the range defined by the standards. This type of
extrapolation should only be applied when prior knowledge of
the relationship is known. Typically this feature is used in
assays where linear relationships are known.
Protocol Menu • 6-53
•
By default, the Min and Max y axis values (for display and
reporting purposes) are determined automatically by KC4.
These values can be overridden by deselecting Auto and
manually entering the Min and Max values.
Defining Extrapolation
The extrapolation factor range must be between 1 and 3.
Using a factor of 1 implies that the defined upper and lower standards are the
limits for concentration interpretation. However, given some assays, the
upper and lower limits may be extended, in this case tentative extension of
the range may be imposed.
•
Example: Assuming a linear x axis we wish to extend the upper
and lower limit by 20%. The lower limit denoted by the first
standard is 50 units. The upper limit denoted by the last
standard is 150 units. The dynamic range therefore is 150-50 =
100 units. Imposing the 20% extension means that the new
range will be: 100 x 1.2 equals 120 units.
This represents an increase in the dynamic of 10 units at the
lower limit and 10 units at the upper limit. Therefore the new
lower limit will be 50-10 equals 40 units. The new upper limit
will be 150 + 10 equals 160 units.
•
Notes: Sample values that fall into the new limits will be
calculated. Those that fall outside these limits will be assigned
> than or < than, respectively. Extrapolation cannot be used for
the Spline routine nor the Point-to-Point fit. These algorithms
cannot calculate values outside the upper and lower defined
standard points.
6-54 • Protocol Menu
User's Guide to KC4
Curve Fit
Use the Protocol|Curves|Curve Fit dialog to select the method by which the
standard curve will be generated. The default setting is Linear Regression.
It is important that the appropriate curve fit is applied to the data otherwise
the results may be invalid. Whichever curve fitting routine is used, it is
essential to check the shape of the curve prior to evaluating data. It is
recommended that the curve is always printed on the report.
The curve fit methods are described below.
Point to Point
Linear interpolation between standard points.
•
The minimum number of standards is 2.
Linear Regression
Linear regression y = ax + b.
y represents unknown response
x represents concentration
a represents the slope of the linear regression
b represents the intercept to the y axis
User's Guide to KC4
Protocol Menu • 6-55
•
The linear regression fit uses the least squares technique. The
better the quality of the fit the more the absolute value of R
tends to 1. This type of curve fitting technique can be used
when you think the data will fall in a predictable linear pattern.
It may be necessary to transform the x and/or y components
using Log axes.
•
Click the Options button to toggle the averaging of standard
replicates. The default is Average Replicates.
Polynomial Regression
This type of regression fitting is an extension of the linear regression
equations.
y= an Xn + an-1 Xn-1+......a1 X + a0 n represents the degrees of the
polynomial regression.
Notes: The degree must be less than or equal to the number of standards
minus 2. The unknown concentrations are calculated by using an
approximate calculation method, linear interpolation between 200 points
evenly spaced on the x axis.
Spline with Smoothing
Spline fitting with a smoothing factor. The curve will appear smoother as
the smoothing factor is increased. The zero factor will force the curve fit
through all data points.
•
The minimal number of standards for the spline fit is 4.
Note: The unknown concentrations are calculated using an approximate
calculation method, linear interpolation between 200 points evenly spaced
on the x axis.
4 Parameter logistic fit
a - d
y = ---------- + d
(1+(x/c)b)
a = (theoretical) response at concentration = 0
b = measure of slope of curve at its inflection point
c = value of x at inflection point
d = (theoretical) response at infinite concentration
x = concentration
y = response (OD)
6-56 • Protocol Menu
User's Guide to KC4
•
It is recommended that:
¾at least one standard is not far from each asymptote.
¾at least 2 standards fall within the linear area of the curve,
one either side of the inflection point.
•
The minimum number of standards is 4.
Curve Interpolation
Select Protocol|Curves|Curve Interpolation to define Y-values for which a
concentration will be calculated.
User's Guide to KC4
•
Enter up to 12 interpolation values in the Y column.
•
Interpolation results can be viewed in Biograph, and can be
printed in an Interpolation report through KC4.
Protocol Menu • 6-57
Validation
Validation is a set of determinant criteria used to assess the suitability of
calibrators for the inclusion in subsequent calculations. Validation critieria,
or Conditions, are expressions based on threshold limits previously
determined from known standardized conditions.
Select Protocol|Validation (with the Protocol Menu in Advanced Mode) to
define Validation Sets and Conditions.
6-58 • Protocol Menu
•
Before defining validation criteria, ensure that all identifiers
have been selected and defined under Protocol|Plate Layout.
•
Define up to 5 different Validation Sets, each containing up to
10 Conditions.
•
The Data Type setting represents the set of data to which the
Conditions equations will be applied.
•
The Conditions fields allow fixed thresholds and/or algebraic
formulas to be used in validating data to satisfy known
conditions of the defined assay.
User's Guide to KC4
A Condition typically combines well identifiers (see Plate
Layout) with the following symbols:
<
less than
<=
less than or equal to
>
greater than
>=
greater than or equal to
Example 1
Conditions to be met:
1) During the first of several kinetic readings, the mean value
of STD1 must fall between 0.200 and 0.300 OD, and the
mean value of STD2 must fall between, 0.900 and 1.300
OD.
2) For each kinetic reading point, the mean value of POS1 must
be less than the mean value of POS2.
Example 2
Condition to be met:
1) The concentration mean of every sample replicate group
must fall between 10.000 and 50.000.
The pound sign # is a generic symbol that can be used to
represent all indexes of a particular identifier.
User's Guide to KC4
Protocol Menu • 6-59
Cutoffs
User-defined Cutoffs are used to screen selected data into defined categories
based on fixed or variable threshold limits. During data reduction, results
are evaluated against the cutoff criteria and each well is assigned a userdefined Symbol.
Select Protocol|Cutoffs (with the Protocol Menu in Advanced Mode) to
defined Cutoffs and Symbols.
•
Before defining cutoff criteria, ensure that all identifiers have
been selected and defined under Protocol|Plate Layout.
•
The Data to Interpret setting represents the set of data to which
the cutoff equations will be applied.
•
The Cutoffs and Symbols fields are interdependent.
¾A Cutoff can be a simple numeric value (1.500), a well
identifier (POS1), or a formula combining the two
(STD1*0.8).
¾Cutoffs should be entered in ascending order.
¾Up to 40 Cutoff equations can be defined.
¾The number of Symbols must exceed the number of Cutoffs
by one.
¾Each Symbol can contain up to 8 alphanumeric characters.
Common symbols include Neg, Eqv, Pos, -, +.
6-60 • Protocol Menu
User's Guide to KC4
Example 1
In the simplified example below, symbols will be applied to
wells as follows:
¾NEG if the OD in the well is less than 1.000.
¾EQV if the OD in the well is greater than or equal to 1.000
and less than 2.000.
¾POS if the OD in the well is greater than or equal to 2.000.
Example 2
In the example below, symbols will be applied to wells as
follows:
¾Neg if the concentration in the well is less than the STD1
mean multiplied by 0.8.
¾Eqv- if the concentration in the well is greater than or equal
to STD1*0.8 and less than the STD2 mean.
¾Eqv+ if the concentration in the well is greater than or equal
to STD2 and less than the STD3 mean multiplied by 1.2.
¾Pos if the concentration in the well is greater than or equal
to STD3*1.2.
User's Guide to KC4
Protocol Menu • 6-61
Reports
Select Protocol|Reports to customize reports for the open protocol:
•
Check Header and enter the text to appear at the top of every
report.
•
Check Protocol Description and Assay Description to include
this information on reports.
•
The Available Data window displays the data and information
available for reporting. Available Data selectable items are
dependent upon the type of protocol selected, as well as the
defined Reading Parameters.
¾Select an individual item by clicking it with the left mouse
button.
¾To select groups of like items, hold the Ctrl key down while
highlighting them.
You can drag and drop
selected items between the
two lists.
¾Click the Add>> button to transfer the item(s) to the bottom
of the Selected Data list.
¾Click the Insert>> button to insert item(s) into the Selected
Data list. Highlight the item in the Report field above
which the inserted item should appear.
¾The Form Feed option forces a page break between reports.
6-62 • Protocol Menu
User's Guide to KC4
Log Report
If Log Report is selected, a warning message(s) will appear on reports when
a special operation has been performed by the user, or if a special event
occurs during the calculations.
Such events/operations include:
•
Use of the Mask function, to mask the value of a well.
•
Use of the Change function, to change the value of a well.
•
Modification of the protocol parameters at run-time, when
using the Biograph module.
•
Failure during the calculation of a transformation.
•
Failure during the calculation of the standard curve.
•
Failure during the calculation of the cut-offs.
Kinetic Curves Report
The Kinetic Curves report shows the kinetic curve in every well, in a plate
format. For example:
User's Guide to KC4
Protocol Menu • 6-63
Well Zoom Report
Each Well Zoom report contains the kinetic curve for one well.
•
To specify a Well Zoom report, highlight Well Zoom in the
Available Data box, enter the desired Wells for kinetic zoom,
then click the Add button.
•
Up to 8 kinetic wells can be overlaid in a single graph. Select
the Wells for kinetic zoom, then click Overlay Wells.
Validation Report
The Validation report contains every validation condition defined for the
protocol and its resulting status.
•
The possible status values are Verified, Not Verified, and Could
Not Be Evaluated.
6-64 • Protocol Menu
User's Guide to KC4
Cutoffs Report
The Cutoffs report lists the defined cutoffs and their calculated results.
•
In the example above, cutoffs were based on the
Concentrations.
Sample Name List Report
The Sample Name List contains all sample names defined for the plate.
Sample name list
#
Name
1
Anderson
2
Ayers
3
Bertrand
4
Buckley
5
Davis
6
Farrell
Plate vs. Table Format
Depending on the nature of the item chosen under Available Data,
information can be reported either in Plate format or in Table format.
Plate Format
•
User's Guide to KC4
With the Plate format selected, data will print in a matrix format
corresponding with the plate layout.
Protocol Menu • 6-65
•
Data for individual kinetic reads can be printed in the Plate
format. Select the type of data (e.g., Meas. OD, Delta OD), then
set the range of reads to include in the report using the From #
and To # fields.
Table Format
•
The Table format is available for kinetic and scanned data. All
kinetic or scanned readings for one or multiple wells. Set the
range of wells to include in the report using the From and To
fields.
Statistics Report
Statistical expansion of the data can be reported. This information is
specifically related to the Identifiers, e.g. controls, standards or samples.
•
6-66 • Protocol Menu
To specify a Statistics report, select an item from the Available
Data box, click the Controls, Standards, or Samples radio
button in the Statistics box, then click the Add button.
User's Guide to KC4
Export
Select Protocol|Export to select exporting parameters for the open protocol:
•
Use the Export dialog to define the format of the export results
file.
•
Check Protocol Description and Assay Description to include
this information in the file.
•
The Available Data window displays the data and information
available for exporting. Available Data selectable items are
dependent upon the type of protocol selected, as well as the
defined Reading Parameters.
¾Select an individual item by clicking it with the left mouse
button.
¾To select groups of like items, hold the Ctrl key down while
highlighting them.
¾Click the Add>> button to transfer the item(s) to the bottom
of the Selected Data list.
¾Click the Insert>> button to insert item(s) into the Selected
Data list. Highlight the item in the Report field above
which the inserted item should appear.
•
User's Guide to KC4
See System Menu, Setup for information on text delimiters.
Protocol Menu • 6-67
Protocol Options
Select Protocol|Options to automate the save, append, report, and/or export
procedures.
•
Select Automatic Save after reading to save the results file
when reading is complete:
¾Enter the Save Directory in which to store the data file.
Click Browse to search for and select the desired location.
¾Select Prompt for Name to manually enter the data file after
reading is complete.
¾Select Auto Name to let KC4 automatically names the data
file using the date and a sequence number. This ensures a
unique file name each time.
Note: The Use barcode as
filename feature is
inactive in KC4 version
2.6.
¾If the reader has a barcode input option, select Use barcode
as filename to automatically save the data file using the
barcode name. If the barcode name contains more than 8
characters, only the last 8 characters will be used. This
feature is not available for all readers equipped with
barcodes.
¾Select a File of Type setting of KC4 Data or KC4
Data+Protocol.
6-68 • Protocol Menu
User's Guide to KC4
•
Select Automatic Append to Kinetic File after reading to
append the current data to an existing data file.
¾This feature allows you to perform several End Point reads
on one plate. You can temporarily remove the plate to
incubate it externally, or perhaps to add reagents.
¾Enter the target directory and file name in the Kinetic File
Name field. Click the Browse button to search for and
select the desired file.
¾Automatic Append to Kinetic File is not available if the
protocol specifies the pre-reading of a blank plate, or
pathlength correction.
•
Select Automatic Print after reading to automatically print
reports when the plate read is complete.
¾Ensure that report parameters are properly set under
Protocol|Reports.
•
Select Automatic Export after reading to automatically export
plate data to a *.TXT file when the read is complete.
¾Ensure that export parameters are properly set under
Protocol|Export.
¾Enter the Export Directory in which to store the text file.
Click Browse to search for and select the desired location.
¾If Automatic Save after reading is selected, the export file
name will match the manually entered file name, the
automatically generated file name, or the barcode,
depending on the automatic save parameters, and will have a
.txt extension.
¾If Automatic Save after reading is not selected, the export
file name will be NONAME.TXT.
¾Select the export text file delimiter under System|Setup.
User's Guide to KC4
Protocol Menu • 6-69
6-70 • Protocol Menu
User's Guide to KC4
Display Menu
The Display menu allows you to check the verification status of your data
and/or to activate the Biograph module to view, adjust, and otherwise
interact with test results as displayed on a curve or curve set. In addition, the
viewing of the Toolbar is controlled through this menu.
Display Menu Quick Reference
The following table provides a brief explanation of each of the options on
the Display menu.
Item
Description
Notes
Curve(s)
Run Biograph. When
Biograph is loaded,
every standard curve
associated with the
current plate appears in
a window.
In the Biograph module, it is
possible to change the curve fitting
method or to delete standard point.
To quit Biograph and to validate
modifications (curve fitting, point
deletion), select File|Validate
And Update KC4.
Validation
Results
Check the status of
every validation
condition.
The possible values for this status
are as follows:
• VERIFIED
• NOT VERIFIED
• COULD NOT BE
EVALUATED: This may occur
when, for example, a condition
refers to an identifier whose
values have all been canceled.
Toolbar
User's Guide to KC4
Show or hide the
Toolbar.
The Toolbar can be moved and
resized. To return the Toolbar to
its original location, simply “drag”
it to the top of KC4's main screen.
Display Menu • 7-1
Curve(s)
Selecting Display|Curves displays the generated graphics associated with
the procedure. This option is active only if the protocol defines a standard
curve or dilution curves.
When the Biograph module is loaded, every curve calculated for the current
plate appears in a window. In the Biograph module, it is possible to change
the curve fitting method or to delete standard points.
To quit Biograph and to validate modifications (curve fitting, point
deletion...), select Validate and Update KC4 from the Biograph File menu.
7-2 • Display Menu
User's Guide to KC4
Validation Results
Selecting Display|Validation Results displays the status of the defined
Validation conditions.
•
If a validation procedure fails, then the results are suspect. The
responsibility for any masking or editing of the data lies with the
user.
•
Validation Status
VERIFIED: The condition is verified.
NOT VERIFIED: The condition is not verified and results are
suspect.
COULD NOT BE EVALUATED: The condition could not be
evaluated. This may occur if, for example, a condition refers to
an identifier with masked replicate values.
User's Guide to KC4
Display Menu • 7-3
7-4 • Display Menu
User's Guide to KC4
System Menu
The System menu allows you to select or change the current reader, and to
set up readers for use with KC4. In addition, the KC4 system administrator
(SuperUser) can use this menu to set passwords and certain system
parameters. The System menu is shown below:
User's Guide to KC4
System Menu • 8-1
System Menu Quick Reference
The following table provides a brief explanation of the options available on
the System menu.
Item
Description
Notes
Readers
Change or setup the
current reader
Select the new reader in the
Available readers list and click the
Current reader button. Setup of
filter/wavelength choice, port, and so
on.
SuperUser LogIn
Enter the SuperUser
mode
If a SuperUser password has been
defined, the access to this mode is
password protected. The SuperUser
mode allows the user to have access
to functions that may be not available
for normal users
SuperUser Logout
Quit SuperUser mode.
Available only when SuperUser
mode is activated.
Change SuperUser Change SuperUser
password.
Password
Available only when SuperUser
mode is activated.
Define Protected
Functions
Functions that can be protected are as
follow:
This option is used to
define functions that
must be available only
for the SuperUser.
• Modify a protocol created by the
SuperUser
• Modify the value of a well
• Mask the value of a well
8-2 • System Menu
Setup
Set up global parameters
(time formats for
kinetics). Set the
character be used as a
separator in exported or
imported files.
Plate Format
Allows user to create and FL600-specific. For more
modify plate formats, and information, refer to the FL600
section of Equipment Interfaces.
to specify a default
format for new protocols.
Reader Control
Sends individual
commands to the reader
(open/close door, temp,
etc)
Not available for all readers. Controls
individual functions including the
incubator temperature and to bring
the plate in and out.
Diagnostics
Run the reader Optics
Test or Universal Plate
Test.
KC4 automatically saves the results
of each test in a database.
Select from KC4’s list or enter any
single character as a custom
separator.
User's Guide to KC4
Reader Selection
Select System|Readers to select or change the current reader.
•
Choose a reader from the Available Readers list, then define the
Configuration (FL600 only), Port, and Filters/Wavelengths
parameters.
•
Click Configuration to define FL600-specific configuration
parameters such as Probe Size.
•
Click Port to define communication parameters including Port,
Transmission Speed, Data Bits, Stop Bits, and Parity.
•
Click Filters/Wavelengths to set up the software
filter/wavelength table.
•
When all parameters have been set, click the Current Reader
button. KC4 will attempt to communicate with the attached
reader.
¾If communication fails, KC4 will provide an informative
error message. Follow the instructions provided in the
message, then try again.
User's Guide to KC4
System Menu • 8-3
FL600 Configuration
This dialog is only applicable to the FL600 reader.
•
Consult the FL600 Order Entry Form that was shipped with the
unit to determine which probes are installed. If this information
cannot be found, note the reader serial number then contact
Bio-Tek Instruments.
•
If necessary, change the Top and/or Bottom Probe Size by
choosing the appropriate option from the pull-down menu.
Note: Once the probe sizes have been defined in KC4, they
need not be re-defined unless a probe with a different size is
installed.
Port Selection
The Port Selection dialog is used to define communication parameters.
8-4 • System Menu
•
Click the COM radio button corresponding with the
communication port through which the computer is connected to
the reader.
•
Click Setup to define additional communication parameters.
User's Guide to KC4
It is the user's
responsibility to define
correct parameters,
otherwise communication
errors will likely occur.
Ensure the communication
settings match those shown
on the reader. These can
usually be found via the
reader's menu system; for
assistance, consult the
reader's user manual.
•
Important! Exercise caution when altering the default
communication parameters. Certain readers (e.g., the
FL600), require a specific setup.
•
Transmission speed: Values commonly accepted by
readers are between 1200- 9600.
•
Data Bits: Options range from 4-8 bits.
•
Parity: Options include No, Odd, Even and Mark.
•
Stop bits: Options include 1, 1.5 and 2 bits.
Filters/Wavelengths
The Wavelengths dialog is used to ensure that the KC4 wavelength table
setup matches that of the attached Bio-Tek reader.
The parameters available
within this dialog are
reader-dependent.
User's Guide to KC4
System Menu • 8-5
•
Note: It is essential that you conform to the specific Bio-Tek
reader procedures for altering the configuration. It is your
responsibility to ensure that correct filters are allocated to their
respective positions otherwise incorrect data will result.
•
The currently selected Reader name and COM port are
displayed at the top of the dialog.
•
Enter the reading Wavelengths in fields 1-6. The Wavelengths
defined here must match those on the reader. See
Filters/Wavelengths (FL600) if using a FL600.
•
If the reader is connected to the computer, click Get
Wavelengths to retrieve the reader's wavelength settings and
store them in KC4.
•
If supported by the attached reader, clicking Send Wavelengths
downloads the wavelengths from KC4 into the reader's internal
wavelength table. The reader then automatically performs a
system self-test.
Filters/Wavelengths (FL600)
Important! In order to obtain accurate results, the KC4 Wavelengths table
must be set up to exactly match the filters installed in the FL600.
KC4 cannot automatically retrieve the wavelength information from the
FL600; this information must be entered manually. Consult the FL600
Order Entry Form that was shipped with the unit to determine which filters
were originally installed. If this information cannot be found, note the
reader serial number then contact Bio-Tek Instruments.
If the exact filter setup is unknown, you can remove the FL600's shroud to
examine the filters in the Excitation and Emission filter wheels. The filters
should be imprinted with the wavelength and bandwidth. Refer to the FL600
Hardware Guide for instructions.
8-6 • System Menu
User's Guide to KC4
•
A - F represent the 6 filter positions on the Excitation and
Emission filter wheels.
•
Excitation refers to the light directed at the sample in the well.
•
Emission refers to the light emitted by the sample in the well.
•
Center is the center wavelength, in nanometers, of the installed
filter.
•
Bandwidth is the filter bandpass width, in nanometers (nm), of
the installed filter. Absorbance filters have a standard bandpass
of 10nm; KC4 stores this information internally and it cannot be
changed by the user. Example: The Excitation/Center setting of
485/20 refers to a center wavelength of 485 nm with a the total
bandpass of 20 nm. This combination will transmit light from
475 to 495 nm (10 nm on either side of the center).
•
A check in the Absorb. column informs the software that the
corresponding filter is an absorbance filter. If at least one
Absorb. box is checked, the 'HOLE' notation appears in the
Center field for Excitation position F.
Note: It is not advised to check Absorb. for fluorescence filters
because the bandpass is not fixed at 8 to 10 nm but can be as
wide as 50 nm or more. Some fluorescence filters or narrower
bandpasses, however, might be able to function as absorbance
filters.
•
A check in the Lum. column informs the software that the
corresponding filter will be used for luminescence reading. If at
least one Lum. box is checked, the 'PLUG' notation appears in
the Center field for the Excitation position E.
Note: When a Lum. box is checked, its corresponding
Bandwidth is not disabled, although the bandwidth is not used
during luminescence plate reading.
User's Guide to KC4
System Menu • 8-7
SuperUser Login
Select System|SuperUser Login to gain entry to the SuperUser mode.
The SuperUser mode allows the system administrator access to options that
may not be available to the average user.
Once logged on as a SuperUser, the System|Change SuperUser Password
and Define Protected Functions become accessible.
Note: When KC4 is installed, there is no SuperUser password defined,
therefore access to the Change SuperUser Password and Define Protected
Functions options is not prohibited. After a SuperUser password is defined,
correct password entry is required for access to these functions.
Change SuperUser Password
Once logged in as the SuperUser, select System|Change SuperUser
Password to change this administration-level system password.
•
•
If the SuperUser password has already been defined then you
must re-enter it before a new password can be defined.
Note: Make sure you remember the password. If you do
forget it, the only solution is to reinstall KC4.
8-8 • System Menu
User's Guide to KC4
Define Protected Functions
Once logged in as the SuperUser, select System|Define Protected
Functions to define the functions that should only be available to the
SuperUser.
•
Mask a value: Allows only the SuperUser to mask (exclude)
individual wells from further analysis.
•
Change a value: Allows only the SuperUser to change plate
data such as Optical Density (OD) or Fluorescence Units (FU).
•
Modify a protocol created by SuperUser: Allows only the
SuperUser to modify protocols created by the SuperUser.
User's Guide to KC4
System Menu • 8-9
Setup
The System|Setup dialog is used to define various global parameters.
•
Select from three fixed Import/Export Separators (semi-colon,
comma, or tab) that will be used in all import and export file
formats. Or specify a custom separator (such as | ) in the Other
field.
¾When plate data is exported, this symbol separates data
items. For example:
[Plate: Meas. OD - blank]
Blank=1.4507
;1;2;3;4;5;6;7;8;9;10;11;12
A;-1.4467;0.2403;-0.8707;0.9753;
¾Before importing a plate data file, ensure that the selected
Import/Export Separator matches what is used in the file.
•
8-10 • System Menu
KC4 accepts the following Time Formats for Kinetics:
Format
Description
HH:MM:SS
Allows for normal kinetics to be meaningfully
reported.
HHH.MM
Allows for long term kinetics to be meaningfully
reported.
SSSS.sss
Allows for medium period kinetics to be reported in
seconds and milliseconds.
User's Guide to KC4
SSSSSSSS
Allows for medium period kinetics to be reported in
seconds.
Sssssssss
Allows for fast kinetics to be reported in
milliseconds.
Note: When involved in calculations such as Transformations,
Cutoffs and Validation conditions, time values are always
expressed in milliseconds, regardless of the time formats chosen
in the System|Setup menu. If you need to use other units,
define a transformation to make the conversion. When time
values are used on the Y axis of a Standard Curve, they are
always expressed in seconds.
•
•
Select an option for the Weighting of Standard Deviation:
Selection
Description
N-1
Used in calculations where the data is assumed to
represent a sample from a larger population.
N
Used in calculations where the data is assumed to
represent the total population from which the
sample was taken.
Choose whether or not to use out of range values in statistics.
Out of range values are those values reports as '<xxxx' or
'>xxxx').
Important! Care must be taken when using this option, as it can
lead to biased results, particularly for standard deviation and
CV. As a warning, statistic results arising out of the use of this
option are displayed in italic in the report.
Example:
SPL1 has three replicates reported with the following
concentrations:
well A1 : 10.230
well A2 : <10
(out of range value)
well A3 : 10.450
User's Guide to KC4
•
If the Use out of range values in statistics option is not
selected, the software will not use concentration of well A2
in the statistic calculations. Therefore the mean, standard
deviation and CV of SPL1 will be 10.34, 0.155, 1.5%
respectively.
•
If the Use out of range values in statistics option is
selected, concentration of well A2 is taken into account (the
value of 10 is used in the statistic calculations). Therefore
the mean, standard deviation and CV of SPL1 will be
reported as <10.22, 0.225, 2.2%.
System Menu • 8-11
Plate Format
System|Plate Format is available only when the FL600 is the current
reader.
Important! In order to obtain accurate results, the appropriate Plate Format
must be defined prior to microplate reading.
Applying specifically to the FL600, the Plate Format database stores
descriptions and dimensions for a wide variety of microplates. This
dimension information is utilized by the FL600 to guide probe movement, so
that readings are taken in the precise microwell locations.
The user specifies which Plate Format will be used within each Protocol. In
addition, the user can specify a default Plate Format that appears in the
Reading parameters dialog when creating new Protocols.
To add, modify, delete, or specify default Plate Formats, ensure that the
FL600 is selected as the current reader, then select System|Plate Formats.
8-12 • System Menu
•
The List of Plates box lists all Plate Formats in the database.
•
The Default Plate field displays the currently selected default
Plate Format.
•
To create a new Plate Format, click the New button.
•
To modify an existing Plate Format, highlight the desired
format, then click the Modify button. Exercise caution if
modifying an existing plate format; measurements must be
precise to ensure accurate results. Consider creating a new
format instead.
User's Guide to KC4
The unit of measure for
plate dimensions is
microns.
Plate Format parameters are described below:
•
Name typically stores descriptive, unique text appropriate for
the microplate, such as 'Corning 96 well plate'.
•
Manufacturer stores the name of the microplate manufacturer.
•
Catalogue # stores the microplate identification number.
•
Length is the longest dimension of the plasticware (the x axis).
•
Width is the shorter dimension of the plasticware (the y axis).
•
Top Left X is the distance from the left side of the plate to the
center of well A1.
•
Top Left Y is the distance from the top of the plate to the center
of well A1.
•
Bottom Right X is the distance from the left side of the plate to
the center of the last well on the plate. In the case of a 96-well
plate, this would be H12.
•
Bottom Right Y is the distance from the top of the plate to the
center of the last well on the plate. In the case of a 96-well
plate, this would be H12.
•
Nb Columns is the number of vertical columns of wells when
viewing the plate in its normal orientation. In the case of a
96-well plate, this would be 12.
•
Nb Rows is the number of horizontal rows of wells when
viewing the plate in its normal orientation. In the case of a
96-well plate, this would be 8.
•
User's Guide to KC4
Well Diameter is the diameter of any typical well.
System Menu • 8-13
Microplate Measurement
If the Plate Format database does not contain a format for a particular
microplate that will be used in the FL600, it is recommended that the plate
be measured and a new format added to the database.
The following diagram demonstrates the proper method for taking
measurements for a 96-well plate. Use calipers, with precision to 0.01 um,
to gather the following measurements. A standard ruler is not precise
enough.
Top Left X
Diameter
Top Left Y
Width
Bottom
Right Y
Bottom Right X
8-14 • System Menu
Length
User's Guide to KC4
Reader Control
If supported by the current reader, Reader Control will be available under
the System menu. This feature can be used to control the reader door or
carrier, and to control incubation.
Door/Carrier
Although this dialog is available regardless of the reader type selected, its
functionality is reader-dependent. The attached reader must have robotic
capability or must be a FL600.
•
Select Plate In to move the plate carrier into the plate reading
chamber and close the door (if applicable).
•
Select Plate Out to move the plate carrier out of the plate
reading chamber and open the door (if applicable).
Pre-Heating
Use the Pre-Heating dialog to:
User's Guide to KC4
•
Set the reading chamber temperature.
•
Turn the incubation unit on or off.
•
Set a timer to “beep” after a specified duration, or when the
requested temperature has been reached.
System Menu • 8-15
•
The Requested field contains the setpoint temperature, in
Celsius. To set the temperature, enter the temperature in the
Requested field, then check On to begin pre-heating.
•
On enables the incubation unit. The temperature will rise or fall
to the Requested temperature, as appropriate.
•
Off disables the incubation unit. The temperature will fall to
ambient.
8-16 • System Menu
•
The Actual field reports the current temperature (Celsius) of the
incubation unit.
•
For the FL600, the Ambient field reports the current
temperature of the outside environment, via a sensor located
within the reader (refer to the FL600 user manual for sensor
location information).
•
The Total Time field reports the total time elapsed since the On
box was checked.
•
Associated with the Total Time field, the Beep after checkbox
and field can be enabled to request that KC4 "beep"
continuously after a specified duration. The duration begins
when the incubation unit is turned on.
•
The Time since reached field reports the total time elapsed
since the incubation temperature has reached the Requested
temperature.
•
Associated with the Time since reached field, the Beep after
checkbox and field can be enabled to request that KC4 "beep"
continuously after a specified duration. The duration begins
when the incubation temperature reaches the Requested
temperature.
•
The REACHED indication appears when the incubation
temperature has reached the Requested temperature.
User's Guide to KC4
As the temperature of the incubation unit approaches the Requested
temperature, it may take a few minutes to settle within an acceptable
tolerance (+/- 0.5° C). During this settling period, the REACHED indication
may appear for a few seconds, disappear, then reappear moments later. The
Time since reached field will automatically reset to 00:00:00 each time the
REACHED indication appears. If the Beep after checkbox associated with
Time since reached field is enabled and set to, for example, 10 minutes,
KC4 will "beep" 10 minutes after the incubation temperature has
consistently settled within +/- 0.5° C of the Requested temperature.
Note:
Diagnostics
The Optics Test and Universal Plate Test should be performed routinely to
validate and document the consistency of a reader's optical and electronic
performance, alignment, accuracy, reproducibility, and linearity.
Optics Test
The Optics Test validates and documents the consistency of reader optical
and electronic performance. For most readers, this test can be performed
from within KC4.
To perform an Optics Test for all readers except the EL311 and the FL600:
User's Guide to KC4
•
Ensure that the reader is properly connected to the computer,
and that all communication parameters are set correctly.
•
Select System|Diagnostics|Run Optics Test. The Run Optics
Test dialog will appear.
•
Enter the reader serial number, then click Start to run the test.
System Menu • 8-17
•
When the test is complete, the Identification dialog will appear.
¾Enter the From, Company, Telephone, Fax Number, and
Comments as appropriate.
¾Click OK to continue. KC4 will display the results. Test
results are saved in a database in the KC4 folder.
Optics Test History
Following each Optics Test, KC4 saves the test results in a database
(CALIB.MDB), located in the KC4 folder.
To view historical Optics Test results:
•
Select Setup|Diagnostics|Optics Test History. The Optic Test
History dialog will appear:
¾To view a list of tests for a specific reader, click the Reader
drop-down arrow.
¾To view a list of tests for a specific operator, click the
Operator drop-down arrow.
8-18 • System Menu
User's Guide to KC4
¾To view test results, highlight the desired date/time from the
list then click View.
¾To permanently delete test results from the database,
highlight the desired date/time from the list, then click
Delete.
Optics Test (FL600)
To perform an Optics Test with the FL600:
•
Ensure that the reader is properly connected to the computer,
and that all communication parameters are set correctly.
•
Select System|Diagnostics|Optics Test.
•
The FL600 Reader Diagnostics dialog will appear. Click Start
to run the test.
•
As each individual test completes, its pass/fail status appears on
the display.
•
When the tests are complete, the results will resemble the
following:
¾Click View Report to see a complete listing of commands
exchanged between KC4 and the FL600 during the
diagnostic procedures. This report can be printed and faxed
to Bio-Tek for help with technical support.
¾Click Close to close the dialog and return to the main
display. Test results are automatically saved in a database in
the KC4 folder.
User's Guide to KC4
System Menu • 8-19
Universal Plate Test
The Universal Plate Test utilizes Bio-Tek's Universal Calibration Test Plate
to validate and document the consistency of reader alignment, accuracy,
reproducibility, and linearity. For most readers, this test can be performed
from within KC4.
Every Bio-Tek Universal Test Plate comes with a “Calibration Test Plate
data sheet.” The data sheet lists the expected absorbance reading for each
location in the test plate at several different wavelengths. This data must be
entered into KC4 before the Universal Test Plate can be used.
To define Universal Test Plates:
•
Select Setup|Diagnostics|Define Universal Plates. The
Define Universal Test Plates dialog will appear, listing the
currently defined test plates by serial number.
•
To modify an existing plate, highlight the desired serial number
then click Modify, or to add a new plate, click Add. The Edit
Universal Plate dialog will appear.
¾If this is a new plate, select the Plate Type then enter its
Serial Number.
¾The top row of the wavelength grid shows eight unique
wavelengths for use with the Universal Plate Test. The
default wavelengths correspond with the calibration test
plate data sheet.
(Optional) To modify one or more of these wavelengths
click Wavelength List.
¾Refer to the calibration data sheet that came with the test
plate to determine the expected OD for each test well at each
wavelength.
For each Location (C1, D4, E2, F5, G3, H6), enter the OD
associated with that well at each wavelength.
8-20 • System Menu
User's Guide to KC4
For readers that support spectral scanning, a Peak
Wavelength value is required. Enter the value in nanometers
(nm).
¾Click OK to save the changes and close the dialog, or
Cancel to close the dialog without saving changes.
To run the Universal Plate Test:
•
Ensure that the reader is properly connected to the computer,
and that all communication parameters are set correctly.
•
The reader should be equipped with the necessary filters to
perform the test. Refer to the calibration data table that came
with the test plate to determine the required wavelengths.
•
Select System|Diagnostics|Run Universal Plate Test. The
Run Universal Plate Test dialog will appear:
¾Enter the Serial Number of the reader under test, or click
the drop-down arrow to choose an existing number.
¾Click the Universal Plate drop-down arrow to select the test
plate to be used. If the desired test plate is not in the list,
select Setup|Diagnostics|Define Universal Plate to define
a new test plate.
User's Guide to KC4
System Menu • 8-21
¾If supported by the reader, the Peak Absorbance Search
option is available. If the box is checked, the Peak
Absorbance Search will be performed in addition to the
standard Universal Plate Test.
¾Choose the wavelength(s) to be included in the test by
clicking the required cells in the Test (Yes/No) column.
Clicking in a cell toggles Yes/No.
Note: If “N/A” appears for a wavelength that you want to
test, update the reader's filter/wavelength table to include the
wavelength.
¾Enter the Operator name, or click the drop-down arrow to
choose an existing name.
¾Enter Comments pertaining to this plate test (optional).
¾When all information has been entered, click Run Test to
begin.
•
Following the directions provided on the screen for loading and
turning the test plate.
•
When the test is complete, KC4 will display the results. Test
results are automatically saved in the CALIB.MDB database in
the KC4 folder.
Universal Plate Test History
Following each Universal Plate Test, KC4 saves the test results in a database
(CALIB.MDB), located in the KC4 folder.
To view historical Universal Plate Test results:
•
Select Setup|Diagnostics|Universal Plate Test History. The
Universal Plate Test History dialog will appear:
¾To view a list of tests for a specific reader, click the Reader
drop-down arrow.
8-22 • System Menu
User's Guide to KC4
¾To view a list of tests for a specific operator, click the
Operator drop-down arrow.
¾To view test results, highlight the desired date/time from the
list then click View.
¾To permanently delete test results from the database,
highlight the desired date/time from the list, then click
Delete.
User's Guide to KC4
System Menu • 8-23
8-24 • System Menu
User's Guide to KC4
Results
Overview
When plate reading is complete, or after data has been entered manually or
read from a file, KC4 displays the plate results, as shown below:
User's Guide to KC4
•
Click the down arrow button associated with the Data field to
select another type of data to view.
•
Generally, if a well contains '?????', no value could be
calculated for it.
•
If a well contains 'OVRFLW' (FL600 only), the well is saturated.
Wells containing OVRFLW are excluded from transformation
equations and statistical calculations.
Results • 9-1
•
To change one or more raw data values, click Change then
change the values in the desired well(s). Click OK to recalculate
and validate results based on the changes.
Note: The SuperUser can protect the use of this function.
Masking is allowed within
most results data sets.
Only those wells masked
within a raw data set (such
as M#1 405) will be saved
as “masked” when the
plate is saved.
•
To mask one or more wells to temporarily exclude them from
calculations, click Mask then click on the desired well(s). Click
OK to recalculate and validate results without the masked
well(s). Note: Masking wells may significantly change or even
invalidate the results. The SuperUser can protect the use of this
function.
¾To unmask a well, click Mask, click on the well, then click
OK. Results will automatically recalculate.
•
To copy data to another application, highlight the desired wells
then select Copy from the Edit menu. Switch to the target
application and select a Paste option.
•
Click Statistics to view statistical information for the identifiers
assigned to this plate.
Data
The available data sets depend on the defined protocol parameters.
To select a data set to view, click
the desired title.
•
to open the Data menu, then click on
The availability of options within this menu is protocoldependent.
¾Template displays the identifiers assigned to each well, with
standard and dilution values if applicable.
¾M 977 (SP) and M 900 (SP) contain the values of the Sample
Plate read at 977nm and 900nm, respectively (necessary for
pathlength correction calculation). See “Pathlength
Correction” on page 6-16 for more information.
9-2 • Results
User's Guide to KC4
¾Measurement numbers (M#1, M#2, M#3) correspond with
the wavelength selection order in the Reading parameters
dialog.
¾A set resembling M#1 405 represents the raw Sample Plate
values obtained at the specified wavelength.
¾A set resembling M#1 405 Blank represents the difference in
readings between the Sample Plate and Blank Plate at the
specified wavelength.
¾A set resembling M#1 405 Corr. represents the final
corrected values after Blank Plate Subtraction, Blank Wells
Subtraction, and/or Pathlength Correction have been
applied. See “Raw Data Correction” on page 6-25 for more
information.
¾Delta OD typically represents the difference in readings
between two specified wavelengths. Note: If the reader
supports >2 wavelengths per read, select Protocol|Multi
Plate Transformations to see how Delta OD is calculated.
¾Concentrations represent the calculated concentration
values in a quantitative assay. Note: Click the Curves
Toolbar icon to see the Data to Interpolate setting, as well
as other curve-fitting parameters.
¾Conc./Dil. represents final concentrations calculated by
multiplying the interpolated concentrations by any dilution
factor imposed on the concentration
¾If Cutoffs are defined in the protocol, select Symbols to see
the cutoff symbol assigned to each well.
User's Guide to KC4
Results • 9-3
Transformation Results
Transformed data is calculated using the formulas defined within the
Protocol|Single Plate Transformation or Protocol|Multi Plate
Transformation dialogs.
•
The symbol ????? is assigned to wells in which the calculation
of the transformation has failed (ex: division by 0, log of a
negative value, etc.).
Calculated Concentrations
Concentrations are calculated by interpolating on the standard curve the data
designated as Data to Interpolate in the Protocol|Curves|Description
menu.
•
9-4 • Results
Values outside the range of the standard curve are designated as
'<lowest conc.' or '>highest conc.', where the 'lowest conc.'
and 'highest conc.' represent respectively the minimum and
maximum concentrations plotted on the standard curve.
User's Guide to KC4
Refer to the System|Setup menu for using such 'out of range
values' in statistical calculations.
Note: These two values can be different from the minimum and
maximum standard concentrations if an extrapolation factor is
applied.
•
To calculate values outside the normal limits of the curve, select
Protocol|Curves|Axes to increase the Extrapolation Factor.
•
The symbol ????? is assigned to wells whose values to
interpolate are not defined and for which it is therefore
impossible to calculate the concentration.
Concentrations x Dil.
This data set reports final concentrations calculated by multiplying the
interpolated concentrations by any dilution factor imposed on the
concentration.
User's Guide to KC4
Results • 9-5
Symbols
If cutoffs are defined in the protocol, each well is assigned a Symbol
representing the cutoff category under which the well falls. The data set
used to calculate cutoffs can be modified under Protocol|Cutoffs.
9-6 • Results
•
Each well should contain its assigned cutoff symbol.
•
If a well contains 'OUT+' or 'OUT-' and cutoffs are based on
concentrations, the well fell outside the range of the standard
curve.
•
Click the Cutoffs button to see the cutoff criteria and the
calculated cutoff value(s).
User's Guide to KC4
Statistics
From within any results window, click the Statistics button to view the
statistical data associated with the results.
•
To view statistics for a different identifier set, click Samples,
Controls, or Standards.
•
The available columns are protocol-dependent.
¾Name: This column contains the name of the sample
associated with the current well.
¾Conc.\Dil.: Concentration or dilution of the current well.
¾Well: Coordinates of the current well.
¾Nb: Represents the number of replicates that were used to
calculate statistics.
¾Mean: Mean of the replicate group.
¾Std Dev: Standard deviation of the replicate group.
¾CV (%): Coefficient of variation of the replicate group, i.e.
the standard deviation expressed as a percentage of the
mean.
User's Guide to KC4
Results • 9-7
Viewing Kinetic Data
KC4 provides several options for viewing kinetic data. The default view is
Kinetic Curves.
9-8 • Results
•
Each well displays a graph of the data points corresponding to
the OD readings taken over time.
•
Hold down the Ctrl key and click on up to 8 wells to overlay the
kinetic curves in one display.
•
Click the Scales button to adjust the data points displayed for
each well. The current scaling parameters are shown in the
lower left corner of the Kinetic Curves display.
•
Click the Calculations button to alter the calculation zone for
kinetic calculations for all wells.
•
Click on a well to enlarge the view of its kinetic curve:
User's Guide to KC4
¾The individual kinetic curve dialog can display up to 8
overlaid kinetic curves.
¾Kinetic calculations such as Max V, Coeff Reg, and Delta t
at Max V, associated with the currently selected plot are
displayed. Click on the down arrow associated with
Selected Curve to choose a different curve.
¾Moving the mouse cursor over a kinetic data point in the
currently selected curve will display coordinates for that
point. Data displayed can include Read #, OD, and
Reading Time.
¾Click on an individual data point to temporarily exclude it
from further calculations, then click the Recalculate button
to exclude the point from kinetic calculations.
Important! Modified data may adversely affect the final
results.
¾To restore an excluded data point, click on the point then
click Recalculate.
¾Click the Options button to adjust the Kinetic Analysis
parameters for the curves shown in this dialog. Refer to
Kinetic Analysis under “Protocol Reading Parameters” in
the Protocol Menu section of this guide for more
information.
Viewing Spectral Scan Data
The default view for spectral scan data is Spectrum Curves:
•
User's Guide to KC4
Each well displays a graph of the data points corresponding to
the OD readings taken across a wavelength range.
Results • 9-9
•
Hold down the Ctrl key and click on up to 8 wells to overlay the
spectrum curves in one display.
•
Click the Scales button to adjust the data points displayed for
each well. The current scaling parameters are shown in the
lower left corner of the Spectrum Curves display.
•
Click the Calculations button to alter the calculation zone for
spectrum calculations for all wells.
•
Click on a well to enlarge the view of its spectrum curve.
If the data point appears
to be “missing” for a
particular wavelength,
this means that the OD
read at that wavelength
is overrange.
¾The spectrum curve initially contains all ODs read at all
wavelengths as defined in the protocol.
¾Spectrum calculations such as Min OD/Max OD and W at
Min OD/Max OD, associated with the currently selected
plot are displayed. Click on the down arrow associated with
Selected Curve to choose a different curve.
¾Moving the mouse cursor over a data point in the currently
selected curve will display coordinates for that point. Data
displayed can include Wavelength #, OD, and Wavelength
value in nanometers.
¾Click on an individual data point to temporarily exclude it
from further calculations, then click the Recalculate button
to exclude the point from calculations.
Important! Modified data may adversely affect the final
results.
¾To restore an excluded data point, click on the point then
click Recalculate.
¾Click the Options button to adjust the Spectrum Analysis
parameters for the curves shown in this dialog.
9-10 • Results
User's Guide to KC4
Viewing Scanned Data (FL600)
The default view for endpoint scanning assays is Mean F.U. (or Mean
OD).
•
Each well contains the mean value of all readings taken across
the well.
•
From the Data pull-down menu, select Std. Dev. F.U./OD or CV
F.U./OD to view additional data.
•
Click on a well to zoom in on it.
•
Picture Mode is a graphical representation of all readings taken
across the well. Move the mouse cursor to any location in a
microwell to see its OD/F.U. values, as well as its X/Y location
in relation to the entire microplate. Click Data Mode to view
the actual fluorescence unit or optical density values taken
across the well.
•
User's Guide to KC4
To exclude/restore a point from the scanning calculations, click
on the desired point, then click Recalculate.
Results • 9-11
9-12 • Results
User's Guide to KC4
Biograph
Overview
Use the Biograph module for curve fitting and manipulation. Biograph can
be used either as a stand-alone product or in conjunction with KC4.
•
To launch Biograph, select Biograph.exe from within the KC4
folder. It can also be accessed via the Windows Programs menu.
•
To start Biograph from within KC4, select Display|Curves to
open Biograph and display the curve(s) currently in memory.
Any changes made in Biograph can be communicated back to
KC4 and saved.
Both KC4 and Biograph allow you to select the mathematical curve that best
fits the standards, and this curve is used for calculating unknowns. The
standard curve is displayed with its formula and the calculated coefficients
and correlation parameters.
The Biograph functions are described in the following sections.
User's Guide to KC4
Biograph • 10-1
File Menu
The File Menu is used to access curve files and data.
File Menu Quick Reference
Item
Description
Notes
New
Create/Define a new
curve.
This option allows you to manually
create a curve by entering the
coordinates of the standard points.
• The 'x' column contains standard
abscises.
• The 'y1', 'y2', … 'y12' columns
contain the ordinate values of
standard points.
• The 'M y' and 'SD y' columns
contain the mean and standard
deviation, respectively, of the y
values. Values in these columns
are automatically calculated and
cannot be edited.
10-2 • Biograph
Open…
Opens an existing
curve file.
Print Setup…
Define print setup
parameters.
Select the printer, printing orientation,
and paper size.
<Recent File>
Lists the most recently
opened curve file(s).
Lists up to 4 previously-opened curve
files, in chronological order of their
opening.
Validate and
Update KC4
Saves changes to the
curve and to
information for the
curve in KC4.
Closes Biograph and returns user to
KC4.
Exit
Exit Biograph.
Returns user to the program from
which Biograph was launched (KC4
or Windows operating system).
User's Guide to KC4
New
Toolbar
Keys:
CTRL+N
Selecting New defines a new standard curve by manually entering the
standard coordinates of the standard points (See also Graph|Standards
menu).
•
The column named 'x' contains standard abscises.
•
The columns named 'y1', 'y2', …, 'y12' contain the ordinate values of
standard points (replicate values.
•
The columns named 'M y' and SD y' respectively contain means and
standard deviations of the y values. Values in these columns are
automatically calculated and cannot be edited.
Save
Selecting Save saves Biograph files in a unique format (extension .GPH).
Toolbar
Keys:
CTRL+S
Note: When a curve has been saved, it is possible to reuse it to process a
plate that does not have its own standard curve (See KC4 module,
Protocol|Curve Description).
Open
Use the Open menu to retrieve previously stored graphic files (with
extension .GPH) and manipulate them in the same manner as newly created
graphs. The retrieved graphic file will be displayed in its own window.
User's Guide to KC4
Biograph • 10-3
Save As, Close, Print Preview, Print
These options are standard Windows options. These options are described
in the Data Menu section of the KC4 Reference Guide.
Validate and Update KC4
If Biograph was launched from within KC4, and if changes are made to the
currently open graph, selecting File|Validate and Update KC4 will close
Biograph and then update the file data in KC4.
View Menu
The View Menu allows the user to customize Biograph by turning on or
shutting off, as required, the status bar and the tool bar.
View Menu Quick Reference
10-4 • Biograph
Item
Description
Notes
Toolbar
Hides or displays the
Toolbar at the top of
the screen.
A checkmark indicates that the
Toolbar is active.
Status Bar
Hides or displays the
A checkmark indicates that the Status
Status Bar at the
Bar is active.
bottom of the Biograph
screen.
User's Guide to KC4
Graph Menu
The Graph Menu allows the user to set and customize the curves that
display in Biograph based on KC4 data.
Graph Menu Quick Reference
Item
Description
Notes
Standards…
Allows the user to
modify the standard
values, unless the
Biograph module has
been launched from
KC4.
• When Biograph is launched from
KC4, standard values cannot be
modified.
• The Mask option allows the user to
delete a standard or a replicate. It
is possible to protect this function
with a password.
Axes…
Allows the user to
The logarithmic scale is invalid if one
select a linear or
of the standards has a negative or null
logarithmic scale in the coordinate.
axes.
Fit…
Allows you to select
the default curve fit
routine.
Five Fit methods are available:
• Point-to-Point: Linear
interpolation between standards.
• Linear Regression: Linear
regression.
• Polynomial Regression:
Polynomial regression 1-6 degree.
• Spline: Spline fitting with
smoothing factor. The curve is all
the more smoothed as the
smoothing factor increases.
• 4 Parameter: 4-parameter logistic
fit.
Overlaying…
Recalculate
User's Guide to KC4
This option is used to
select curves to be
overlaid in a single
graph.
• It is possible to overlay up to 8
different curves.
This option is used to
recalculate the curve
when a standard point
or replicate has been
deleted.
(none)
• If some values have been entered in
the Interpolations window,
intercept lines are displayed for
every curve.
Biograph • 10-5
Standards
The Standards dialog allows you to enter or modify the values of standards.
Note: When Biograph is started from KC4, values of standards can not be
modified.
•
x: Represents the known concentrations of the x axis.
•
M y: Represents the Mean y variable. This field cannot be
entered and is calculated automatically.
•
SD y: Represents the Standard Deviation of the y variable.
This field cannot be entered and is calculated automatically.
•
y1 to y12: Represents the response values to be plotted
(replicates).
Data Entry
Use the cursor keys to move through the matrix. Entry of the x
concentration and response y values must be concluded before clicking OK
or pressing the Enter key.
The Mean y and SD y will automatically be calculated for the data.
After the x and y variables have been accepted then the graphic will be
generated. You can modify the axes, curve fit and impose interpolations on
the graphic. In addition you can edit the graphic.
Editing Standards
You can edit the standard data by either:
10-6 • Biograph
•
Using the Mask feature. This temporarily suspends the data
value. Click on the value to be suspended.
•
Changing the data value or eliminating the data value.
User's Guide to KC4
Recalculation will automatically be performed on the data after you press the
OK button.
Note: You can use the KC4 Protection functions to prevent the
masking/changing of data values. See System|SuperUser Login|Define
Protected Functions for more information.
Axes, Fit, Interpolations
These features function similarly to functions of the same name in KC4. See
KC4 module, Protocol|Curve menu for more information.
Overlaying Graphs
Biograph allows you to create and edit multiple graphs in the same session.
•
The graphs must be open before they can be overlaid.
•
Up to 8 graphs can be overlaid, regardless of curve fit type, but
all must have the same axes (either all linear or all logarithmic).
The curve overlay will display in a new window labeled
OVERLAYED CURVES.
User's Guide to KC4
Biograph • 10-7
•
The curves in this window are not editable, unlike the individual
curve windows. Nor can the interpolations in this window be
adjusted directly (you must adjust in the individual curve
windows, if necessary).
However, any changes which you make to an individual curve or
curves are reflected immediately in the "shadow curve" in the
OVERLAYED CURVES window once you have calculated the
change using Graph|Recalculate.
•
Note: To overlay several graphs, all desired graphs must be
open in Biograph.
•
10-8 • Biograph
After Overlaying, you can impose new Interpolations on the
composite graphic.
User's Guide to KC4
Curve Window
The replicate of each data point is displayed with a green square and the
mean replicate value is displayed in a red circle.
•
Click on an individual data point to temporarily exclude it from
further calculations, then select Graph|Recalculate button to
recalculate the curve without the point.
Important! Modified data may adversely affect the final results.
•
To restore an excluded data point, click on the point then select
Graph|Recalculate.
•
User's Guide to KC4
Select File|Print to print the graph.
Biograph • 10-9
10-10 • Biograph
User's Guide to KC4
Appendix A
Using KC4 in a Robotic System
KC4 has an OLE function which allows external robotic applications to
interface with Bio-Tek automated microplate readers for reading parameter
control and data collection. Typically, the robotic application software will
retrieve a list of user-defined KC4 protocols to execute the reading process,
with KC4 running in the ‘background’.
When the robotic reading process is complete, the user is free to open KC4
to recall collected data files and perform further data reduction.
To use KC4 in a robotic system, the user must first define typical protocols
as explained in the KC4 User’s Guide. The robotic application will instruct
KC4 to execute the protocol as defined. Data that is collected will be stored
in a user-specified directory. KC4 can be operated separately from the
robotic system interface for routine data reduction.
Limitations of KC4 in a robotic system are as follows:
•
The user cannot access KC4 while under Robotic Control.
•
A user cannot start a robotic run with KC4 running. The
application must be closed before beginning a run.
•
Only the following Data Reduction functions are supported:
¾Kinetic Analysis Data (e.g., Max V, Mean V, etc.).
¾Scanning Analysis Data (e.g., Mean OD, Std Deviation, CV
in each well for FL600 applications).
¾Spectrum Analysis Data (e.g., Min/Max OD, Wavelength at
Min/Max OD).
•
User's Guide to KC4
If the Export Data capability is required (or if Auto Export is
enabled within the protocol), the export parameters in the
protocol must be limited to the following:
Appendix A, Using KC4 in a Robotic System • 11-1
¾Template
¾Meas OD or Delta OD (in the case of a dual wavelength
read), or MeasOD1 to Meas OD(n) (in the case of a multiwavelength read).
¾Kinetic Analysis Data (e.g., Max V, Mean V, etc.).
¾Scanning Analysis Data (e.g., Mean OD, Std Deviation, CV
in each well for FL600 applications).
¾Spectrum Analysis Data (e.g., Min/Max OD, Wavelength at
Min/Max OD).
11-2 • Appendix A, Using KC4 in a Robotic System
User's Guide to KC4
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