XF 24 Extracellular Flux Analyzer Manual

e
XF 24 Extracellular Flux Analyzer
Manual
Part no. 102609-400RevA
First Edition:
102609-400 Rev A December 2012
Revision history:
Revision A: first release January 2013
Copyright:
Copyright ©2013 Seahorse Bioscience Inc. All rights reserved.
Trademark Information:
Microsoft, Windows and Excel are registered trademarks of Microsoft Corporation. All
other trade names are properties of their respective companies.
Seahorse Bioscience Inc.
16 Esquire Road
N. Billerica, MA 01862
(800) 671-0633
(978) 671-1611 Fax
Submit technical support questions or comments about our documentation to:
support@seahorsebio.com
For the latest information on products, visit our web site at:
www.seahorsebio.com
Printed in U.S.A.
3
General Assay Flow Chart for XF Assays
4
Table of Contents
Table of Contents
4
Introduction
1.
Seahorse Bioscience XF Technology Overview
5
2.
Technical Specifications
6
3.
XF Instrument and Controller Installation Procedure
7
Unpacking and Component identification
Suitable Locations for the instrument
Removing the Shipping Cartridge
Internal Components of the XFe24
Set-up and Interconnects: Cable Installation
Safety Considerations
8
9
10
11
12
13
4.
Basic Operation of the XFe24 Instrument
Power and Warm-up
XFe Wave Software and Login
Cleaning and routine maintenance
14
14
15
16
Appendices
I. Troubleshooting
II. Support Contact and Ordering Information
III. XFe Wave Software User Guides
17
17
18
5
Introduction
1. Seahorse Bioscience XFe Instrument Overview and Intended Use
The Seahorse Bioscience XFe analyzer measures the rate of change of dissolved
oxygen and pH in the media immediately surrounding living cells cultured in a
microplate. Changes in the extracellular media are caused by the consumption or
production of analytes by the cells. Therefore, a sensitive measurement of the media
flux can be used to determine rates of cellular metabolism with great precision and in a
totally non-invasive, label-free manner.
A unique feature of the XF technology is its ability to make accurate and repeatable
measurements in as little as five minutes. This is accomplished by isolating an
extremely small volume (less than 3 μl) of media above the cell monolayer. Cellular
metabolism causes rapid, easily measured changes to the “microenvironment” in this
small volume.
Typically, a measurement cycle is performed for 5-8 minutes. The media is gently
mixed and the analyte levels are then measured until the oxygen concentration drops
approximately 20-30% and media pH declines approximately 0.1-0.2 pH units. The
measurement is performed using optical fluorescent biosensors embedded in a
disposable cartridge that is placed into the specially designed Seahorse tissue culture
microplate.
Baseline metabolic rates are typically measured 3-4 times, and are reported in pmol/min
for OCR (Oxygen Consumption Rate) and in mpH/min for ECAR (Extracellular
Acidification Rate). Compound is then added to the media and mixed, and then the
post-treatment OCR and ECAR measurements are made and repeated. As cells shift
metabolic pathways, the relationship between OCR and ECAR changes.
Because XF measurements are non-destructive, cells can be profiled over a period of
minutes, hours or days.
The XFe system includes a bench top analyzer and touch screen controller, disposable
measurement/compound delivery cartridges, as well as microplates for cell culture,
calibration solution and XFe software.
Consumable cartridges, microplates and calibration reagents are packaged and sold in
sets under the trade name “FluxPaks”. One FluxPak contains the materials for 18
assays.
6
2. Technical Specifications
Model
XFe24
Dimensions:
Width x height x depth
Controller:
19” x 17” x 12”
48 cm x 43 cm x 30 cm
Analyzer:
16” x 24”x 17”
41 cm x 61 cm x 43 cm
Weight:
Controller: 22 lbs. / 9 Kg
Analyzer: 45 lbs / 20 Kg
Power Requirements
100-240V AC 50/60Hz
6A Analyzer
3.2A Controller
Power cord rating
3- wire (grounded) AC power cord rated 10A or greater
Power fuse ratings
10A
Environmental
Conditions
“Normal” Environmental conditions- indoor use, altitude to
2000 m
Room Temperature
Range
+64°F - 86°F / +18°C - 30°C
No direct sunlight
Humidity less than 80% for temp up to 31°C, decreasing
linearly to 50% at 40°C
Internal Temperature and
Environment
Controlled to user-selected temperature not less than 10°C
above ambient temp
No CO2 control
Software OS
Windows 7
Data Interface
RS232c (internal)
TCP/IP (external)
USB Type B
7
XFe Instrument and Controller Installation Procedure
**XFe Instruments must be installed by trained
Seahorse Bioscience personnel ONLY.**
Unpacking and Component Identification
The XFe Analyzer system is packaged in two boxes; the optional XF Prep Station is
packaged in one box.
Upon receipt, immediately check each box for damage. Shipping damage must be
reported to the transportation company and Seahorse Bioscience using the contact
information on page (ii) of this document.
The following items are included in an XFe system:
XFe Analyzer- The analyzer is a temperature controlled instrument containing all optical
and electronic measurement components to measure oxygen and proton flux of cells
grown in XF cell culture plates. The analyzer is used in conjunction with XFe sensor
cartridges.
Plate Preparation Station (PS) (Optional) – The PS is recommended to accurately
exchange growth media for assay media in the tissue culture plate wells. It also
contains a temperature-controlled chamber for incubating the cell culture plates in a
37°C/non-CO2 environment after the media exchange and a temperature-controlled
dry bath for warming and incubating the assay media. Refer to the Installation Guide
included with the Plate Preparation Station for safety & operating instructions
pertaining to installation of the PS. NOTE: The Plate Preparation Station must be
installed and connected to the XFe Analyzer by trained Seahorse Bioscience
personnel for optimal performance.
Controller- Operation of the Analyzer is done through a high resolution color LCD touch
screen that may be installed in front of or beside the XFe. The controller
communicates with the analyzer using a single serial cable and a single USB A-B
cable. The controller communicates with the optional PS via a single USB/Serial
adapter cable with a DB9-DB25 Null Modem cable.
8
XFe24 Extracellular Flux Analyzer
The XFe24 and optional plate preparation station require two people to lift and handle safely.
Each person should firmly grasp the base of the unit at opposite ends to one another. Use OSHA
standards for lifting techniques.
The following items are included in the XFe24 instrument and controller boxes
Instrument/Controller
Quantity
XFe Instrument
1
XFe Controller
1
Power Cord (instrument)
1
Power Cord (controller)
1
9
Power Supply (controller)
1
RS232 Cable
1
USB Cable
1
USB Extension Cable
1
Extra Fan Filters
2
Suitable Locations for the XFe system
XFe Analyzers are designed for laboratory use. The internal environment of the
analyzer is controlled to a preset temperature by the user, and therefore, laboratory
room temperature must be maintained within the range listed in the specification table.
Analyzer temperature control performance can be monitored using the status display on
the right side of the analyzer.
The XFe uses optical detection technology to measure extremely low levels of
fluorescent emission from analyte sensors. While the analyzer has been designed to
shield room light, excessive light (such as direct sunlight) should be avoided.
Seahorse Bioscience Personnel will Remove Shipping Protection Components
from the XFe Analyzer During Installation
10
**XFe Instruments must be installed by trained
Seahorse Bioscience personnel ONLY.**
The analyzer is shipped with protection components that must be removed prior to use.
Seahorse Bioscience recommends these components be removed by Seahorse
Bioscience personnel during installation.
To prevent damage during shipping, the instrument is shipped with a cartridge loaded
onto the probe head and lowered onto a plate on the tray. These items must be
removed prior to running the first assay. Seahorse personnel will remove these items
during installation.
11
Internal components of the XFe Analyzers
The measurement enclosure is the heart of the XFe Analyzer. Removing the side doors
reveals the measurement chamber in which the assay is conducted. The electro-optics
hardware is enclosed in a card cage in the rear chamber and this is connected to the
probe head through a set of bundles of fiber optic cables. The base of the enclosure
contains the primary controller board and heater assembly. The following describes
each of the hardware features pointed out in the images below.
1 LCD Message screen—Displays current instrument action and internal
temperature.
2 Cover latches—Pull on indented hand-holds molded into the gull wing doors
(not shown) to lift them up, exposing the internal components of the
instrument
3 Thermal tray
4 Probe Head and Injection Manifold—The probe head consists of 24 “light
pipes” to carry the optical signals to and from the sensors. The Injection
Manifold uses compressed air to inject compounds loaded into Sensor
Cartridge ports
5 Fiber Bundles
6 Front Stripper Arm—Used for sensor cartridge removal at the end of assay
runs
7 Bar Code Reader—Reads bar code on Sensor Cartridge and cell plate
8 Circulating Fan—Helps to stabilize environment inside XFe Analyzer
5
8
4
7
1
3
2 (not
shown
XFe Front/Side View
XFe Front View
6
12
Setup and Interconnects: Cable Installation
The XFe Analyzer is operated from a touch screen computer monitor mounted to a
stand, referred to as a controller. One RS232 cable and one USB cable handle the
communication of commands and data between the instrument and the controller.
The controller may be connected to an external network, as well as additional hardware
such as the optional XF Plate Preparation Station, via the ports on the underside.
Refer
to
the
Technical
Specifications section to identify
each cord, and refer to the
diagrams on the right to identify
the sockets.
1. Connect power cords:
Network
Port
Power Input
One power cord is used to
connect the instrument to a
grounded AC (mains) outlet.
A second power cord is used
to connect the controller
power supply module to the
AC supply.
The power
supply module is then
connected to the socket at
the bottom of the controller.
Refer
to
the
Plate
Preparation
Station
Installation
Manual
for
instructions on connecting
the PS to the XFe controller
and power source.
USB Ports (4)
Serial Port
Controller Ports (underside)
USB Port from
Barcode Reader
Serial Port for
Controller
Connection
XFe Rear Panel
2. Connect the data cables to
the analyzer
One RS232 cable connects
the controller serial port to
the analyzer socket labeled
“COM”
A second cable (USB)
connects the analyzer socket
labeled “USB” to the USB port on the
13
controller directly adjacent to the
network (Ethernet) port. This port
must be used for proper
functioning of the instrument and
barcode reader (see diagram to
right).
A third cable (RS232 serial cable
with a separate USB adaptor)
may be connected to one of the
other USB ports on the controller
if the optional XF Prep Station
will be connected to the
instrument. Refer to the Users’
Manual included with the XF
Prep Station for complete instructions.
3. Connect external network cables. The controller may be networked via the Ethernet
port on the controller.
Safety Considerations
The XFe and optional XF Prep Station require two people to lift and handle safely. Each
person should firmly grasp the base of the unit at opposite ends to one another. Use
OSHA standards for lifting techniques.
Safe operation of the instrument requires that the cover be securely attached and plate
tray door is closed. This also prevents heat loss and system cooling which can affect
data quality.
The door opens automatically when the tray is extended, allowing the operator to insert
or remove the assay consumables. The operator must exercise caution during the
loading of the wellplate/cartridge in order to avoid the possibility of a pinch hazard. After
the wellplate/cartridge is securely on the tray, the operator’s hand must be removed
from the area of the tray before continuing the assay. After the command is given to
continue the assay via the controller, the tray will move slowly back into the instrument
and the door will close.
When the cover is securely attached and tray door is closed, a physical switch is
engaged to monitor the system. An optical sensor is used to determine the status of the
door.
The XFe has a heater that maintains a stable internal system temperature. Typically, the
temperature will be maintained at 37°C, as monitored by temperature sensors and
controllers embedded in the tray and above the tray. Two fans circulate air through the
14
plenum containing the heater. A thermal fuse will disable the heater should it reach an
abnormally high temperature.
4. Basic Operation of the XFe Analyzer
Power and Warm up
To power on the touch screen
controller, press the power switch on
the top of the controller. To prevent
accidental power down of the
controller, the switch may be
disengaged in the “Power Options”
menu of the Windows OS control panel
under the “Advanced” tab.
Turn the instrument on using the power switch on the rear panel
On the right side of the instrument
near the access door, there is an
LCD message screen. When the
instrument is powered up the LCD
message screen will show:
When XFe Wave software is
launched on the controller, the LCD
message screen will update and
show the following display. Note
that it is recommended to allow at
least 2 hours for the instrument to
fully warm and equilibrate to the set
temperature.
Measurements are taken using the appropriate XF Sensor Cartridge for your instrument
(purchased exclusively from Seahorse Biosciences). The XF sensor cartridge has a
single multifluour spot on the bottom of each probe that is sensitive to both oxygen and
proton concentration. By measuring the concentration of each analyte over time, the
oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) may be
calculated.
15
XFe Wave: Login
Launch XFe Wave and sign in. A default user name and password will be provided by
Seahorse personnel during installation; Seahorse recommends changing these
immediately for security. Allow the XFe Analyzer to warm to 37°C for at least two hours.
Ideally, the instrument should be left on overnight to ensure consistent and accurate
temperature. Note that the instrument will NOT warm to 37°C unless the software is
launched (simply turning on the instrument only will NOT engage the heater).
XFe Wave Start Up Screen
Refer to the XFe Wave User Guides (Appendix III) included with the instrument for an
overview of the user interface, as well as operating details of XFe Wave (including
instrument settings, user profiles, defining an assay protocol, running an assay and data
analysis). The XFe Wave User Guides may also be accessed through the Help section
of XFe Wave (touch “Help” from the home screen– indicated below).
16
Refer to the XFe Wave User Guide for details on importing and exporting data files
(including assay templates and data files from instruments from/to third-party software
programs).
Note that XFe Wave may be downloaded and installed on any personal computer via
the Seahorse Bioscience website. The desktop version should not be installed on the
XFe controller, however. Instrument commands may only be given through XFe Wave
installed on the controller.
Additional information and protocols for preparing medium associated with XF assays,
experimental design, running XF assays and analyzing XF data may be found online at
www.seahorsebio.com,
or
by
contacting
Seahorse
Bioscience
Support
(support@seahorsebio.com; 1800-671-0633 or 001-978-671-1600 (option 3))
Cleaning and Routine Maintenance
The XFe instrument was designed for minimal cleaning and maintenance by the user.
Note all consumables are disposable and none of the instrument components come into
contact with the cell plate at any time, preventing cross-contamination of biological or
chemical materials. However, there are some minor tasks that can be performed by the
user if necessary.
Probe Cleaning:
If cell culture plate wells are over-filled, or over a long period of time, the optical surface
on the bottom of the measurement probes may require cleaning. Use a cotton-tipped
stick and 95% ethanol to clean these optical windows beneath each probe. Take care
not to scratch the surface and do not use a cleaner that leaves a residue.
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Air filter replacement:
At approximately one year intervals, the air filter at the rear of the analyzer should be
replaced. The filter retainer snaps away from the housing- use a screwdriver or wooden
stick to remove it.
18
Appendix I - Troubleshooting
Please visit www.seahorsebio.com for FAQs.
For further information, please contact Seahorse Support or your local distributor:
Email: support@seahorsebio.com
Phone (US): 1-800-671-0633 #3
Phone (world-wide): +1-978-671-1600 #3
Appendix II – Contact, Support and Ordering Information
For questions about XF technology, the XFe Analyzer,
Prep Station, XF experimental design, data analysis,
troubleshooting and other information:
Email: support@seahorsebio.com
Phone (USA): (800) 671-0633, option #3
Phone (world-wide): +1 (978) 671-1600, option # 3
To order XFe FluxPaks (including cell culture plates,
sensor cartridges and calibrant solution) or other supplies:
Email: orders@seahorsebio.com
Phone: (978) 671-1600 option #2
Fax Purchase Orders to: (978) 671-1611
Appendix III – XFe Wave Software User Guide
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e
102612-100 Rev D
XF Wave Software User Guide
XFe Wave Software User Guide
TABLE OF CONTENTS
1) Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2) Defining Groups and Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3) Mapping Groups to the Plate Map . . . . . . . . . . . . . . . . . . . . . . . . . . 5
4) Defining a Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5) Analysis: The Overview Tab . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6) Analysis: The Data and Configuration Tab . . . . . . . . . . . . . . . . . . 10
XFe Wave Software User Guide
1) INTRODUCTION
XFe Wave Software, designed for the XFe Extracellular Flux Analyzer, simplifies the design and analysis of XFe experiments. The XFe
Wave Software User Guide highlights key functionality within the software, and offers guidance to users when performing the most
common tasks associated with designing and analyzing extracellular flux experiments. The XFe Wave Software User Guide is divided
into the key sections of the software, based on the major goals performed on each screen. Note: This user guide refers to default tab
and screen names, which can be customized by the user during assay design and analysis.
- Defining Groups and Conditions
- Mapping Groups to the Plate Map
- Defining a Protocol
- Analysis: The Overview Tab
- Analysis: The Data and Configuration Tab
To Start a New Assay
Open the XFe Wave Software by clicking the XFe Wave software icon on your desktop. Select Blank Assay in the My XFe tab and click
he Design button. This will launch a new assay design in the XFe application. Fill out the relevant experimental information in the Getting
Started tab, and move to the Group Definitions tab to begin your experimental design.
1.
XFe Wave Software User Guide
2) DEFINING GROUPS AND CONDITIONS
The Group Definitions tab includes the Assay Conditions and Plate maps screens, where you begin to describe the biological variables
in your experiment. Although the fields provided are not required to be completed in order to perform an XF assay, good record keeping
here can significantly impact your data analysis and interpretation of multiple experiments. Conditions are divided into 4 categories:
Injections, Pretreatments, XF Medium, and Cell Type.
Injections
XF Medium
The injections condition describes the contents of each set of
injections in the assay. Each specific injection strategy
represents the contents of up to 4 injections (through ports A, B,
C, or D). Click on the description tabs within each injection
strategy below (window at right) to describe the contents of each
injection for a specific port. If the entire plate is using the same
injection strategy for all 4 ports, only one injection condition
needs to be entered.
The XF medium condition describes the assay running medium
used during the XF assay. Base medium and supplements for
each different running medium used in the assay (for example,
with or without pyruvate) should be recorded here.
Cell Type
The pretreatment condition describes any treatment the cells have
received prior to the XFe Assay. This can include anything from
compound treatments to differences in growth medium. In this
window, enter the name of the pretreatment condition, and provide
record-keeping details in the text box associated with the field.
The Cell Line condition describes the source of cellular material
used for the XF assay. Information about the cell line, seeding
concentration, and passage number are described in these fields.
102612-401 Rev D
Pretreatments
2.
XFe Wave Software User Guide
2) DEFINING GROUPS AND CONDITIONS
Automatically Generate Groups
Manually Generate Groups
XFe Wave software will automatically generate groups based on
the number of independent variables you define in your assay. If
you only define one variable for a specific condition, it will
assume that this is a global variable, and this information will not
be used to differentiate groups. If more than one variable is
defined, however, the software will automatically calculate the
number of groups, assuming every possible combination of
independent variables.
In cases where a factorial experiment is not desired, you can also
generate groups manually, and select the conditions that are
used to describe the differences between the groups.
Example:
Example:
In this example, the same set of conditions were defined, but if
fewer than nine groups are desired, you can add the specific
number of groups you want by clicking the Add button under the
Well Groups window. Click the Add button 3 times to produce 3
groups, and then define the variables that make up each group
by choosing from a drop down window for each condition and
selecting the appropriate variable.
In the above example, multiple variables were assigned to the
conditions Pretreatments (AG-34565, AG-38474, AG-98789) and
Cell Type (C2C12, Clone 1, and Clone 2), while only one variable
was assigned to Injections and XF Medium. When the Generate
Groups button is clicked, the software automatically generates 9
groups, fulfilling every possible combination of Pretreatments
and Cell Type.
You have now described the Assay in terms of groups, and are
ready to map to the well plate.
3.
XFe Wave Software User Guide
3) MAPPING GROUPS TO THE PLATE MAP
When designing an experiment using XFe Wave software, one of the most critical pieces of information is the Plate Map. This is where the
information about each well in the experiment is defined, and will be used for group statistics and analysis when the assay is complete.
Groups
Distribute Groups
There are a multiple options for determining the number of
groups in your assay. You can determine them using the
“Generate Groups” feature or assign them manually in the
Conditions tab (see User Guide – Conditions), or add groups in
the Plate Map tab. Here we describe the shortest method for
generating groups in the Plate Map tab, using group name as a
differentiator, and mapping the plate directly.
The XFe Wave software will automatically distribute groups
across a plate if desired. This command will determine the
maximum number of replicates automatically and distributes the
groups in vertical sections across the plate.
Example:
Example:
To assign groups to the plate map, click on the group name to
highlight, then click on the individual wells to assign this group to
that well. This can be done individually, or by rows and columns
by selecting the appropriate row and column buttons (labeled
A-H and 1-12) above and to the left of the plate.
Background Wells
The XFe Wave software will automatically default to Seahorse
recommended background wells. The number and locations of
these wells may be changed by clicking on the Background tab
in the Well Groups window, then double-clicking on the desired
well to assign it as a background well.
102612-402 Rev D
In the Well Groups window, click Add 3 times to generate 3
groups. At this point, 3 groups will be visible in the Well Groups
list. To change the name (i.e. from Group 1 to C2C12/Compound
A), double click on the highlighted portion of the group name and
enter the new name. If you have already set up conditions in the
previous windown, choose the appropriate conditions from the
dropdown list, otherwise you can leave these blank.
When the group list has been populated, click on the Distribute
Groups button. This command will automatically fill the plate
vertically, with the maximum number of replicates possible for
the number of groups defined.
4.
4) DEFINING A PROTOCOL
XFe Wave Software User Guide
Once groups and conditions are defined and mapped to the plate, it is time to move on to the Instrument Protocol tab. Here you will
define the assay protocol, including measurement and injection cycles.
When the Instrument Protocol tab is selected,
the first 3 commands of the protocol are
preselected to allow for rapid protocol
definition. Every experiment must begin with a
Calibration, so this will always be the first step in defining a
protocol. The next step Equilibrate – this command is defaulted
as on (box is checked), because Seahorse always recommends
including the equilibration step before the assay begins to
assure temperature stability. In rare cases, equilibration is not
needed, so you have the ability to uncheck this box if desired.
The third default command is a measurement cycle. These are
the first 3 measurments that occur before the first injection
(referred to as Basal Measurements) and since a majority of
assays will begin with these 3 measurements, the command is
added by default.
You may designate a name for this measurement by selecting
the text field at the top of the column and typing in the desired
information (i.e., Basal). Seahorse recommends three
measurment cycles for each rate; these cycles, as well as default
“Mix” and “Measure” times, are pre-populated. Each of these
commands is customizable using the up-and-down arrows, or by
selecting the field and typing in the desired numbers.
The “Mix” command instructs the instrument to raise and lower
the sensor cartridge probes to ensure that analytes (or following
injections, compounds) are uniformly dispersed within the
medium in each well. The “Measure” command instructs the
instrument how long to record the flux of analytes in the transient
microchamber once the sensor cartridge probes are lowered.
Mix and Measure times are customizable, and may need to be
optimized to fit your application. You also have the option of
defining a customized “Wait” time (default Wait settings are 0
minutes). The Wait command instructs the instrument to delay
before taking a measurement.
Injection
Select the Injection button to add an injection cycle into the
protocol.
Each time you select the Injection button, a
new Injection command will be included in the
assay protocol.
The software will automatically select the next available port to
assign the injection to. You can change this default setting by
touching another port (A, B, C, or D). Only available ports will be
selectable – if another is already assigned it will appear greyed
out. When selecting an injection, the software will automatically
add a set of measurement cycles after the injection. Default mix,
wait, and measure times will be included; you may customize
these times, as well as the number of cycles, if required. If a
measurement is not required following an injection, you can
uncheck the “Measure after Injection” box. Touch the Summary
task bar under the measurement cyle box to view the
compounds assigned to these ports.
102612-403 Rev D
Default Commands
5.
XFe Wave Software User Guide
4) DEFINING A PROTOCOL
Custom Sequence
Most XFe experiments will only require measurement and injection
cycles as defined above, but in some user-defined assays, a
custom sequence of commands is required. In these cases, touch
the Sequence button to add a custom sequence cycle to the
assay. Here, you can add as many mix, wait and measure cycles
as your protocol requires, in any order. This command allows for
maximum flexibility of your assay parameters.
Save and Save as Template
When your assay design is
complete, save the file in either of
2 formats.
Choose the standard “Save As”
option if the assay is not one that
you are planning to repeat. You
may begin the assay immediately
if desired and connected to an
instrument. Once the instrument
has completed the protocol, a
results file will be generated for
data analysis.
To come back and reuse some of
the conditions defined in this
assay repeatedly, choose the
“Save as Template” option. By
selecting this option, you may also
begin the assay immediately and
generate a results file for analysis,
however you will also be able to open up this protocol at a later
date, and the fields entered will be pre-populated with the specific
parameters you entered previously.
6.
XFe Wave Software User Guide
5) ANALYSIS: THE OVERVIEW TAB
The Overview tab allows for flexible data analysis within Wave software. This is where kinetic graphs for both OCR and ECAR are
displayed, and common tasks, such as exclusion of outlier wells are performed. Group statistics are also calculated and displayed within
this tab. You can add any number of Overview tabs by clicking the Add View button and choosing Overview from the drop down menu.
The Kinetic Graph
This can be done from any rate measurement, if OCR is selected,
O2 consumption rate data will be displayed, if ECAR is selected,
proton flux data will be displayed. In some cases, it is desirable
to overlay the OCR and ECAR on the same graph. This can be
done by selecting a different item in the Y2: dropdown menu.
This allows for all combinations of data to be displayed.
Further manipulation of the kinetic graph visualization can be
accomplished by selecting the Options button. In this pop-up
window, you may configure specific settings for each axis. The
maximum, minimum, and interval fields allow you to set scale on
each axis. The Thickness field allows for visualization of tick
marks on the graph (1–thinnest, 5–thickest). Error bars can be
toggled on and off by checking the Error Bar box.
The kinetic graph is the most common way to
display data from the XFe Analyzer. The default
view displays time on the X-axis (in minutes) and
rate on the Y-axis. You can change the displayed rate (OCR or
ECAR) by selecting it from the dropdown menu above the graph
(Labeled Rate).
102612-404 Rev D
When viewing a specific rate (such as OCR), the
raw level data can be displayed by selecting
“Level” from the Y1: dropdown menu.
7.
XFe Wave Software User Guide
Rate Details and Bar Graph
In the upper right hand corner of the
Overview tab, the Rate Details grid
is displayed. Here, individual rate
data points are displayed for each
well for a specific measurement
number. To change the
measurement # shown in the Rate
Details grid, choose it from the list
in the dropdown menu labeled
Measurement. The measurement
number selected will be highlighted
in a blue column on the kinetic
graph as a reminder of which rate
data is being displayed in the Rate
Details grid; the measurement
information dictated by the user in
the kinetic chart informs which rate details are displayed in the
grid. If OCR is selected in Rate dropdown menu, OCR will be
displayed in the Rate Details grid. The grid is where individual
wells can be excluded from the kinetic graph and the group
statistics data. Click on a well to exclude it (excluded wells will
show up as shaded wells with a black border. When a well is
excluded it will no longer be shown on the graph, and will also be
excluded from group statistics calculations. Use the column
(numbers) and row (letter) buttons to exclude entire sections of
the grid.
5) ANALYSIS: THE OVERVIEW TAB
These rates are also visualized in Details bar chart found below
the Rate Details grid. Here, the rate data is displayed in a bar
chart, either by group or by well (toggle between the two with the
Display dropdown menu). This useful tool can be used to view
group differences for a particular measurement (group mode), or
to help identify outliers for specific groups (well mode).
Group Details
The final window found in the Overview tab is the Group Details,
found in the lower left side of the screen. This window has two
functions. First, it acts as the legend for both the kinetic graph
and the rate details grid and bar chart. The group colors shown
in these visualizations tie in with the group names listed in the
Group Details window. The second function of this window
allows group statistics to be displayed in this window by clicking
the Details button, which allows the mean and standard deviation
for each group in the experiment to be displayed.
Note: Wells which have been previously excluded in the Rate
Details grid will automatically be excluded from the calculation of
these metrics. Double clicking on the colored square for the
group will prevent the group from being displayed on the kinetic
graph, but will not exclude the wells from the calculations.
Double click the square again to turn this group back on.
8.
XFe Wave Software User Guide
6) ANALYSIS: THE DATA AND CONFIGURATION TAB
The Data and Configuration tab has a number of uses: raw data export, experimental summaries, and instrument settings. Navigate through
the specific areas of the Data and Configuration tab using the list of topics on the left hand side of the screen.
Data
Summary
The Data window is where all of the raw data from your experiment
is listed. The raw data is displayed in three main forms: level data,
rate data, and group data. The fluorescent data and the calibration
data can also be acquired from this view. To view a particular data
set, click on the specific tab within the window (in the following
example, the Rate tab) to show the data in a grid format.
The summary window shows a printable version of the protocol
summary and the group plate map, along with injection plate maps
for each port. This information is used to record how the
experiment was run (mix, wait, and measure times, along with
injection sequences are shown), as well as information about where
the groups were located within the plate.
102612-405 Rev D
Each data point is identified with a well column and a group
column, so the data can be displayed in a number of different
formats. To sort the data by a specific column, click on the column
header (Well, Measurement, etc.), and the data will automatically
be sorted by this column. The data can also be exported to
Microsoft® Excel from this tab. Right click anywhere on the graph
(for touch screen, hold your finger on any part of the grid for 3
seconds). From the dialog box, choose Export, name your file, and
choose a save location. The data grid will be exported exactly as it
appears in the data window; further downstream manipulation can
occur by opening the file in MS Excel.
9.
Corporate Headquarters
European Headquarters
Asia-Pacific Headquarters
Seahorse Bioscience Inc.
16 Esquire Road
North Billerica, MA 01862 US
Phone: 1.978.671.1600 • 800.671.0633
Seahorse Bioscience Europe
Symbion Science Park, Boks 22
Fruebjergvej 3
2100 Copenhagen DK
Seahorse Bioscience Asia
199 Guo Shou Jing Road
Suite 207
Pudong, Shanghai 201203 CN
201212144
www.seahorsebio.com
10.
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