Below you will find brief information for Seahorse XF Analyzer XFe96, Seahorse XF Analyzer XFe24. These analyzers are designed to perform real-time, non-invasive measurement of cellular bioenergetic parameters like oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a microplate format. It allows you to study the effects of drugs, genetic modifications, or environmental changes on cellular metabolism.
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© Agilent Technologies, Inc. 2017
No part of this manual may be reproduced in any form or by any means (including electronic storage and retrieval or translation into a foreign language) without prior agreement and written consent from Agilent Technologies, Inc. as governed by
United States and international copyright laws.
Manual Part Number
S7894-10000
Edition
First edition, June 2017
Printed in USA
Agilent Technologies, Inc.
5301 Stevens Creek Boulevard
Santa Clara, CA 95051 USA
Warranty
The material contained in this document is provided “as is,” and is subject to being changed, without notice, in future editions. Further, to the maximum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a particular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing, use, or performance of this document or of any information contained herein. Should
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Limited Rights as defined in FAR 52.227-14
(June 1987) or DFAR 252.227-7015 (b)(2)
(November 1995), as applicable in any technical data.
Agilent Seahorse Wave software is the experiment design, instrument control, data acquisition, data analysis, and file management software for Agilent
Seahorse XF, XFe, and XFp Analyzers. This user guide provides detailed information on each function in Wave software.
The typical Agilent Seahorse XF workflow can be outlined in three steps:
• Design your experiment
• Acquire data
• Analyze results
Design your experiment
First, design your experiment, called an assay template file. Wave software contains several default assay template files for the standardized Agilent
Seahorse assay kits (for example, XF Cell Mito Stress Test). Use the Agilent
Seahorse default assay templates and customize as necessary. Wave also provides a Blank assay template for those users who prefer to create a new assay template for each experiment.
Wave Desktop is the preferred software for designing or customizing assay templates for Agilent Seahorse XFe and XFp Analyzers, however the same functions can be performed using Wave Controller software (for Agilent
Seahorse XFe Analyzers only). See “Create/Edit Assay Templates" on page 14
more for information on creating/customizing assay templates.
Acquire data
After creating an assay template for the experiment, transfer the template to the XFe Analyzer using a USB flash drive or network drive (active network connection required). If the assay template is created using Wave Controller on the XFe Analyzer, the assay can be performed immediately.
Analyze results
After completing the assay, transfer the assay result file from the XFe or XFp
Analyzer to Wave Desktop for data analysis. Wave Controller automatically saves a backup copy of the assay result file locally on the XFe Controller
(computer).
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 3
Templates
View and organize your assay template files from the Templates view. Use the
Create, Export, Import, and Duplicate buttons to easily manage your list of template files.
Results
View, open, and manage all your assay result files from the Results view. Click the
Options button to configure Favorite Places, file locations where assay result files are stored, such as a network directory or local PC folder.
Catalog
Use the Catalog to store Compounds, Pretreatments, Media, and Cells that are frequently used for template design and modification. Wave provides a prepopulated Catalog of the standardized Agilent Seahorse kit components as well as other reagents, media, and cell types. You can add (and delete) entries within the Catalog view only.
Options
Configure the local Template and Catalog file directories as well as the default
Instrument Protocol times.
Help
Agilent Seahorse Technical Support contact info, software version and auto-compile feature for System Files.
4
Figure 1
Wave home views
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User
The Templates view displays all assay template files saved on the XFe
Controller (Wave Controller) and on your PC (Wave Desktop). (See
.)
An assay template (.asyt) file contains the required information to run an experiment on the XFe and XFp Analyzers - Group Definitions, Plate Map layout, and Instrument Protocol. Assay templates can be reused for the same assay, or customized and saved as a new template for another assay on the
XFe/XFp Analyzer.
Figure 2
Templates display
Assay templates are color coded based on the type of assay template.
Table 1
Assay templates
Template type
Agilent Seahorse
Blank
User-customized
Template color
Blue
White
Green
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 5
Blank template
Agilent Seahorse default template
User-customized template
Figure 3
Assay template color examples
Wave software automatically installs 10 default Agilent Seahorse assay template files, which contain assay-specific information for standard Agilent
Seahorse assays. The blank and Agilent Seahorse templates are not editable; modifications made to a Agilent Seahorse template will be saved as a new assay template (green icon). (See
• Blank
• XF Cell Energy Phenotype Test
• XF Cell Mito Stress Test (Acute Injection)
• XF Glycolysis Stress Test
• XF Glycolysis Stress Test (Acute Injection)
• Dependency - XF Mito Fuel Flex Test
• Flexibility - XF Mito Fuel Flex Test
• XF Buffer Factor Assay
• XF CO
2
Contribution Factor Assay
• XF Glycolytic Rate Assay
• XF Glycolytic Rate Assay (Induced Assay)
6 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User
The Results view displays recently opened assay result files, the corresponding file directory where each assay result file is located, and Favorite Places. (See
.) Opening a new assay result file displays the default analysis view - the Quick View. After modifying and saving an assay result file, Wave automatically displays the last modified analysis view the next time the assay
Results file is opened.
Figure 4
Wave home results
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 7
8 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User
5
1 Design Agilent Seahorse Experiments with Wave
14
17
18
Assign groups automatically 20
21
21
22
23
23
24
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 9
10
24
28
2 Perform Assay on the Agilent Seahorse XFe Analyzer
Transfer Assay Template(s) to an Agilent Seahorse XFe Analyzer 30
Transfer assay template using a USB flash drive 30
Transfer assay template using a network drive
30
Start Your Agilent Seahorse XF Assay 31
Add and Remove Measurement Cycles in Runtime
33
35
XFe Assays at non-37 °C temperatures
35
Required operational and assay guidelines 35
Set alarm (Temperature tolerance range) 38
38
39
40
40
Cartridge barcode read failure 40
Cell plate barcode read failure 41
45
46
Analysis View #3 - OCR vs. ECAR 47
Kinetic graph (rate versus time) 48
Scatter plot (rate 1 versus rate 2)
48
50
51
51
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
51
Introduction to Acidification Data 52
Data Analysis Using the XF Glycolytic Rate Assay Report Generator 57
57
58
58
58
Baseline to a Rate Measurement (%)
60
Baseline to a Control Group (%) 61
Standard Deviation and Standard Error of the Mean 61
Rate Overlay: OCR, ECAR, PER, PPR, O2, or pH
62
63
63
64
66
Modifying Assay Result Files 67
67
68
68
71
74
Export to Agilent Seahorse XF Report Generators
77
80
82
83
Display values for: Average, Count, Maximum, Minimum, or Sum 84
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 11
4 Managing Agilent Seahorse Files
88
Export, duplicate, and remove assay templates
88
Recent assays, recent places, and favorite places
91
92
93
93
94
95
96
Advanced options (Wave controller, Agilent Seahorse XFe analyzers ONLY)
97
99
12 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Agilent Seahorse XFe96 and XFe24 Analyzers For use with Wave Desktop
Wave 2.4 User Guide
Create/Edit Assay Templates 14
Step 3: Instrument Protocol 21
13
1
Design Agilent Seahorse Experiments with Wave
To create a new assay template or modify an existing template:
1
Open
Wave 2.4.
2
3
4
Click
Templates (below Wave Home).
Select the
Blank template, a Agilent Seahorse Default template, or a
User-customized template and click Open or double-click the template icon.
Add, remove, or modify group definitions; and create new assay groups.
(See
“Step 1: Group Definitions” on page 15.)
5
6
Assign groups to the Plate Map. (See
“Step 2: Plate Map” on page 20.)
Review or modify Instrument Protocol. (See
7
8
Save the template, transfer it to XFe/XFp Analyzer. (See “Saving Assay
Run the assay. (See
“Step 4: Run Assay” on page 24.)
14 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Design Agilent Seahorse Experiments with Wave
1
Group definitions is comprised of four components:
• Injection Strategies
• Pretreatments
• Assay media
• Cell type
Figure 5
Group definitions
Injection strategies describe the contents of up to four injections (one for each of the four ports: A, B, C, and D), more than one injection strategy can be performed in an assay. For example, on the same plate, two injection strategies can be defined for the XF Cell Mito and XF Glycolysis Stress Test.
To add one or more injection strategies:
1
Click
Add next to Injection Strategies. (See
.)
2
3
Enter a name for the injection strategy if desired. The default name when adding new injection strategies is Inj. Strategy # (# corresponds to how many injection strategies have already been added).
Next, specify the contents of each injection starting with Port A. Click
Add Compound, and type in a compound name (for example oligomycin), or use the
Compound Catalog drop-down menu to select a compound from the
catalog. (See Figure 6 on page 16.)
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 15
1
Design Agilent Seahorse Experiments with Wave
4
If additional injections are called for from ports B, C, and D, repeat step 3.
Figure 6
Additional injection ports
shows two defined injection strategies for the Agilent Seahorse XF
Cell Mito Stress Test assay for different concentrations of FCCP:
• Mito Stress Test - 1 µM FCCP
• Mito Stress Test - 2 µM FCCP
16
Figure 7
Injection strategies for the Agilent Seahorse XF Cell Mito Stress Test assay
Pretreatments describe a treatment the cells have received prior to performing the assay, such as a genetic manipulation or prolonged exposure to compounds.
To add one or more Pretreatments:
1
2
3
Click
Add next to Pretreatments. (See Figure 8 .)
Enter a name for the pretreatment or use the Pretreatment drop-down menu to select a pretreatment from the catalog. The default name when adding new pretreatments is Pretreatment # (# corresponds to how many pretreatments have already been added).
If desired, specify the type of pretreatment using the
Description field.
displays two Pretreatment conditions, Control and Experimental.
Figure 8
Pretreatment conditions
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Design Agilent Seahorse Experiments with Wave
1
Assay Media describes one or more assay mediums used in the assay. Record the medium and supplements for each medium used in the assay:
To add one or more Assay Medium:
1
Click
.)
2
Enter a name for the assay media, or use the Media drop-down menu to select a media from the catalog. The default name when adding a new assay media is
Assay Media # (# corresponds to how many assay media have already been added). (See
.)
Figure 9
Assay Media
3
Enter additional details if desired for: Source, Supplements, Prepared By, and so forth.
Cell Type describes the biological material/samples used, including information about the cell line, seeding density, and passage number.
To define one or more Cell Types:
1
Click
2
Enter a name for the cell type, or use the Cell Line drop-down menu to select a cell type from the catalog. The default name when adding a new cell type is
Cell Type # (# corresponds to how many cell types have already been
Figure 10 Cell Type
3
If desired, enter additional details for: Seeding Density, Lot, Source, and
Passage number.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 17
1
Design Agilent Seahorse Experiments with Wave
The three dots next to each group definition enable two functions: Delete the group definition entry and Duplicate the group definition entry.
Figure 11 Duplicate and Delete function toggle
After creating the individual Group Definitions, create assay groups, and assign the various conditions to each group based on your experimental design. This can be done automatically by clicking the Generate Groups button.
This function uses the independent Group Definitions to calculate the number of groups, assuming every possible combination of independent conditions. An example of the combinatorial logic using the Group Definitions below shows how this function results in a total of eight unique groups (2 * 2 * 1 * 2 = 8):
• Injection Strategies = 2 (Mito Stress Test - 1 µM FCCP and Mito Stress Test -
2 µM FCCP)
• Pretreatments = 2 (Control and Experimental)
• Assay Media = 1 (Mito Stress Test Assay Medium)
• Cell Type = 2 (C2C12 and HepG2)
Figure 12 Automatically generate groups
18 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Design Agilent Seahorse Experiments with Wave
1
If desired, Assay groups can be created manually:
1
Click
Add Group to create Group 1. (See
Figure 13 Add groups
2
Use the drop-down menus to assign the appropriate Group Definitions to
Figure 14 Duplicate and Delete function toggle
3
Repeat for each manually generated group.
To change the name of the group, double-click the
Group 1 text, or right-click and select Edit. To change the color of the group, use the drop-down color menu next to the group name. After adding Group Definitions and generating groups, assign groups to the Plate Map. (See
“Step 2: Plate Map” on page 20.)
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 19
1
Design Agilent Seahorse Experiments with Wave
The Plate Map displays group assignments on the Cell Plate. Wave uses the group assignments on the Plate Map to calculate group statistics (average rates for each measurement performed and the standard deviation or standard error for each rate measurement) after the assay has completed. There are two ways to assign groups to the Plate Map.
Click Distribute Groups. Wave automatically determines the maximum number of replicates per group, and assigns wells to each group starting with column
1. (See
.)
20
Figure 15 Distribute Groups
1
2
Click the group in the Groups list on the left.
Click the well on the Plate Map to assign that individual well to the selected group. You can assign an entire row or column to a group by clicking the row or column label, or drag-and-drop to select an area of the Plate Map to assign to the selected group. (See
.)
Background wells have default Plate Map coordinates based on the type of XFe or XFp Analyzer. You can modify the coordinates of the background wells or add/remove background wells if desired. Use the steps described in
.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Design Agilent Seahorse Experiments with Wave
1
The Instrument Protocol is the series of commands performed during an assay, the timing of when these commands occur, and the duration. The default
Agilent Seahorse assay templates have a preconfigured Instrument Protocol, and do not require modification.
Every Instrument Protocol includes the following steps:
• Calibrate (always ON)
• Equilibrate (highly recommended for every assay)
• Baseline Measurement cycle
Figure 16 Default protocol commands
Calibrate is always the first step in a protocol and cannot be disabled. The
Calibrate step reads the coefficients of the Sensor Cartridge and Cell Plate to ensure accurate data acquisition.
Equilibration ensures temperature stability before beginning an assay. The default setting for Equilibration is
ON. Equilibrate can be disabled although this is strongly discouraged.
Measurement cycles indicate the steps in the Instrument Protocol when rate data is collected for oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The standard Agilent Seahorse Instrument Protocol consists of three measurement cycles before the first injection (called
Baseline) and three measurement cycles after each port injection. Each measurement cycle consists of three commands: Mix, Wait, and Measure.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 21
1
Design Agilent Seahorse Experiments with Wave
• Mix: The amount of time to raise/lower the sensor cartridge to ensure analytes and drug compounds are uniformly mixed in each well before/after an injection, as well as to reintroduce oxygen to the well after forming the microchamber for each measurement.
• Wait: The amount of time to delay the Measurement step after the Mix step.
This command is required for the XFe24 Analyzers in every assay, but may be added to Instrument Protocol for any analyzer.
• Measure: The amount of time to record the flux of analytes in the transient microchamber once the sensor cartridge probes are lowered following a Mix
(or Wait) command.
The default measurement cycle times are:
• Agilent Seahorse XFe96 and XFp Analyzer: 3 minutes Mix; 0 minutes Wait;
3 minutes Measure
• Agilent Seahorse XFe24 Analyzer: 3 minutes Mix, 2 minutes Wait, 3 minutes
Measure
The Baseline measurement cycle is the starting point for every Agilent
Seahorse assay and consists of three measurements before the first injection.
Data acquired during the Baseline measurement cycle provide valuable information about the bioenergetic status of the cells under starting conditions.
22
Figure 17
Default protocol commands
To add an injection command to the Instrument Protocol, click
Injection. Wave automatically selects the compound injection ports sequentially, therefore, the first time you click
Injection, port A will be assigned to injection 1. A grayed out port indicates it has already been assigned to an injection. (See
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Design Agilent Seahorse Experiments with Wave
1
Figure 18 Measurement information
Wave also automatically adds the measurement cycles after adding an injection. Default
Mix, Wait, and Measure times are instrument-dependent, and can be modified in Instrument Options (Wave Home > Options).
To view and edit the
Mix, Wait, and Measure timing, click the drop-down arrow next to
Edit Measurement Details. (See
Figure 18 .) Use your keyboard to enter
specific
Mix, Wait, and Measure times or adjust the number of Cycles for each measurement. The up and down arrows can also be used. To achieve optimal results, a minimum of three cycles per measurement is recommended. The default Mix, Wait, and Measure times are instrument-dependent, and can be modified in
Instrument Options (Wave Home > Options).
To change the name of each command in the Instrument Protocol, click the text
field at the top of the column. (See Figure 18 .)
Most Agilent Seahorse assays do not require the use of custom cycles, however depending on the type of experiment or your experiment design, a custom cycle may be appropriate. To add a custom cycle command to the
Instrument
Protocol, click Custom. A custom cycle enables multiple Mix and Wait steps without a Measurement. Note that the Agilent Seahorse XF Report Generators are not compatible with Instrument Protocols containing a custom cycle.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 23
1
Design Agilent Seahorse Experiments with Wave
The General Information section provides editable fields for project information, plate information, or other important notes related to the assay.
Any information entered on this view is saved in the assay result file (*.asyr).
Figure 19 Assay summary
All errors or warnings are displayed on the right side of the Run Assay step.
(See
.) Prior to transferring the template to the XFe or XFp Analyzer, correct any errors that are displayed. A typical error message is the notification that some assay wells have not been assigned to a group on the
Plate Map. If a template is designed correctly, Wave displays an All Set
confirmation message. (See Figure 20 .)
24
Figure 20 Error and Warning messages
This feature applies to Wave Controller software only, and requires an active internet connection on the XFe Analyzer. Recipients must be added before starting an assay. Wave Desktop does not email assay result files.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Design Agilent Seahorse Experiments with Wave
1
To add an email address:
1
Open the template file.
2
3
4
On the Run Assay navigation step, click Show Advanced Options.
(See
.)
Type in the email address below
Email Settings, and click Add.
(See
.)
Repeat for each email address.
Figure 21 Advanced Options
Adjust the default
Port Volume values using the up/down arrows, or by typing in a value using your keyboard. This is for notation-purposes only, and does not
affect data calculations. (See Figure 21 .)
Version Information displays the Wave software version along with Agilent
Seahorse XF
File version info (internal-use only). (See
To display a summary view of the Instrument Protocol, click Protocol.
(See
Figure 22 .) To edit the Instrument Protocol, click the
Instrument Protocol navigation step before running the assay on Wave Controller, or saving/transferring the assay template from Wave Desktop to the XFe or XFp
Analyzer.
Figure 22 Protocol
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 25
1
Design Agilent Seahorse Experiments with Wave
To display a summary view of the Group Definitions and Plate Map, click
Figure 23 .) To edit the Group Definitions or Plate Map, click the
Group Definitions or Plate Map navigation step before running the assay on Wave
Controller, or saving/transferring the assay template from Wave Desktop to the
XFe or XFp Analyzer.
Figure 23 Group Summary
Click Port A (B, C, or D) for a summary of each injection from the selected port.
(See Figure 24 on page 27.) The summary information includes:
• Compound concentration
• Name of the solvent (if specified)
• Percentage of solvent used for each compound
• Port volume
To modify the compound name, compound concentration, solvent name or percentage, click the
Group Definitions navigation step, then select the appropriate Injection Strategy to edit before running the assay on Wave
Controller, or saving/transferring the assay template from Wave Desktop to the
XFe or XFp Analyzer.
26 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Design Agilent Seahorse Experiments with Wave
1
Figure 24 Port A summary
To print or save a PDF of the Assay Summary, Protocol, Group, and Port A-D summaries for the assay template, click Print Summary.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 27
1
Design Agilent Seahorse Experiments with Wave
After editing an assay template, click the
Save button to overwrite the template with the newly modified details/content. To create a new template file, and preserve the original template file details, click the
Save As button.
Figure 25 Saving options
Click
Save As to display the Save As Template window (See Figure 26 .):
1
Enter the name of the assay template in the Name field (required).
2
3
4
Enter the Author name (optional).
Enter a description of the assay in the Description field (optional).
Click Save.
Figure 26 Save As Template window
28 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Agilent Seahorse XFe96 and XFe24 Analyzers For use with Wave Desktop
Wave 2.4 User Guide
Transfer Assay Template(s) to an Agilent Seahorse XFe Analyzer 30
Start Your Agilent Seahorse XF Assay 31
Add and Remove Measurement Cycles in Runtime 33
This chapter applies to Wave Controller software for Agilent Seahorse XFe96 and Agilent Seahorse XFe24 Analyzers ONLY. The functions described in this chapter do NOT apply to Wave Desktop or Agilent Seahorse XFp Analyzer software.
Assay template files (.asyt) can be designed using Wave Desktop and Wave
Controller. Assay templates created using Wave Controller can be run immediately after designing the template. Assay templates created using Wave
Desktop must be transferred to Wave Controller to perform an assay. For nonnetworked XFe Analyzers, assay templates must be exported from Wave
Desktop to a USB flash drive, then imported to Wave Controller. If the XFe
Controller has an active network connection, assay template files can be transferred/imported directly through a shared network location.
29
2
Perform Assay on the Agilent Seahorse XFe Analyzer
Several options to import assay templates to Wave Controller are outlined below.
3
4
1
2
Save template to a USB flash drive.
Plug the flash drive into the USB port on the Agilent Seahorse XFe
Controller.
Select the USB flash drive, and locate the template file(s) (.asyt).
Double-click the assay template to automatically import to Wave Controller software.
5
6
3
4
1
2
Save template to a USB flash drive.
Plug the flash drive into the USB port on the Agilent Seahorse XFe
Controller.
Click
Templates.
Click
Import.
Select the USB flash drive, and locate the template file (.asyt).
Select one or multiple template files on the USB flash drive, then click
Open.
3
4
1
2
Power ON the XFe Controller (if OFF).
Start Wave Controller software.
Locate the assay template file (.asyt) in the network directory.
Double-click the assay template to automatically import to Wave Controller.
3
4
1
2
5
6
Power ON the XFe Controller (if OFF).
Start
Wave Controller software.
Click
Templates.
Click
Import.
Locate the template file (.asyt) in the network directory.
Select one or multiple template files on the network drive, then click
Open.
30 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Perform Assay on the Agilent Seahorse XFe Analyzer
2
C A U T I O N
After customizing or transferring an assay template to Wave Controller, click
Start Run to begin the XFe assay.
1
2
3
Click
Start Run, and select a location to save the assay template file. The assay result file will be saved in the same location following assay completion. Assay template and result files can be saved to a USB Flash
Drive, shared network directory, or locally on the XFe Controller. Wave
Controller will also automatically save a backup assay result file locally on the XFe Controller in the location: C:\ProgramData\ Seahorse Bioscience,
Inc\Seahorse Wave\Assays.
After selecting a save location, the tray door on the XFe Analyzer will open.
Place the Sensor Cartridge (hydrated and loaded with compounds) along with the Utility Plate onto the tray. Ensure the Cartridge fits properly on the Utility Plate, the lid is removed from the Cartridge, and the direction of the Cartridge is in the proper orientation.
After loading the Sensor Cartridge and Utility Plate, press
I’m Ready to initiate Sensor Cartridge Calibration. The time to complete Calibration for assays at 37 °C is approximately 10-20 minutes. For assays performed at temperatures other than 37 °C, an additional 30 minutes of PreCalibration time will be added to ensure accurate data acquisition.
Figure 27 Load Calibrant Utility Plate prompt
4
After completing Calibration, Wave Controller will display the Load Cell
Plate message prompt. Click Open Tray to eject the Utility Plate and load the
Cell Plate. Ensure the lid is removed from the Cell Plate before loading.
Figure 28 Load Cell Plate prompt
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 31
2
Perform Assay on the Agilent Seahorse XFe Analyzer
5
Click Load Cell Plate to initiate Equilibration. Once Equilibration has finished, the assay will begin acquiring the first Baseline measurement.
Figure 29 Load Cell Plate prompt
6
After completing the final Instrument Protocol step, the assay will finish and display an Unload Sensor Cartridge message prompt. To eject the
Sensor Cartridge and Cell Plate from the XFe Analyzer, press
Eject.
Figure 30 Unload Sensor Cartridge prompt
7
The assay result file will automatically save to the location specified prior to starting the assay, and can be opened immediately following the assay using the on-screen Assay Complete prompt.
Figure 31 Assay Complete prompt
32 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Perform Assay on the Agilent Seahorse XFe Analyzer
2
Figure 32 Real-time measurement cycle data
During a Agilent Seahorse XF assay, data is acquired, calculated, and presented in the real-time. For certain assays, it may be necessary to add or remove measurement cycles in real-time.
Wave Controller 2.4 enables real-time Instrument Protocol modification for commands that have not yet been executed. To display the Instrument Protocol for the selected command, click on an Instrument Protocol step above the data.
(See
Figure 33 Selected command instrument protocol
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 33
2
Perform Assay on the Agilent Seahorse XFe Analyzer
Click Edit Measurement Cycles to display the Edit Number of Cycles window.
(See
Figure 34 .) Use the up/down arrows (or manually type a number) to
add/remove measurement cycles from the selected command.
Figure 34 Edit Number of Cycles
34 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Perform Assay on the Agilent Seahorse XFe Analyzer
2
The Widget icons are located on the lower left side of Wave Controller software, and display the status XFe Analyzer, current temperature, and controls to eject/insert the tray and raise/lower probes. t Status widget: Connection status between the XFe
Controller (computer), Wave Controller (software), and the XFe Analyzer.
Temperature widget: Current tray temperature and heater status display.
Tray widget: Manually eject or insert tray, with or without a Utility Plate or Cell Plate.
Probes widget: Manually unload a Sensor
Cartridge and raise/lower the probes.
Agilent Seahorse XFe Analyzers have been validated to deliver desired target temperatures in the range of 16-42 °C provided the ambient room temperature is 12-20 °C below the target temperature, and in the validated operational room temperature range of 4-30 °C. To understand the relationship between
the sample temperature and ambient temperature, see Figure 35 on page 36
for the temperature chart.
• For all non-37 °C operation, the XFe Analyzer must equilibrate overnight in required ambient temperature.
• If required to set up the XFe Analyzer in cold room, avoid direct fan sources. (See
• For all non-37 °C operation, the tray heater must remain ON, do NOT turn the tray heater OFF.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 35
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• For assay temperatures below 30 °C, hydrate the Sensor Cartridge in the dark at room temperature.
• Prior to starting an assay, an additional 30 minutes of precalibration equilibration time has been added to ensure temperature stability.
36
Figure 35 Temperature chart
To adjust the Target Temperature (set point) using the up/down arrows, click on the
temperature (12-20 °C above ambient). Refer to the temperature chart shown in
.
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N O T E
Figure 36 Temperature widget
Changing the Target Temperature requires OVERNIGHT equilibration to the new set point.
Figure 37 Tray Heater window
Other Temperature Widget functions are:
• Turn the heater ON/OFF.
• Set the tolerance range for temperature fluctuation. If the temperature is above or below the acceptable tolerance range from the temperature set point, the Temperature Widget will change color (
), and the Status
Indicator light (top of the XFe Analyzer) will change from blue to amber.
For networked XFe Controllers, Wave Controller software will automatically send an email notification to specified recipients.
Figure 38 Temperature widget color change
To save any changes on the Tray Temperature window, click Save.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 37
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Figure 39 Temperature Alarm
To set the alarm:
1
2
Check the Alarm On box in the Tray Temperature window.
Click Save.
Disable the alarm by unchecking the
Alarm On box, then click Save. If the Tray
Temperature exceeds the Tolerance Range and the alarm is activated, click
Reset Alarm to acknowledge and reset the Tray Temperature alarm.
To ensure the Tray Temperature starts within the Tolerance Range, check the current temperature of the XFe Analyzer before beginning an assay. For any suspected temperature issues or unexpected temperature fluctuations, contact
Agilent Seahorse Products Technical Support.
38
Figure 40 Tray control widget
Use the
Tray Control Widget to manually eject a Utility Plate or a Cell Plate in
XFe Analyzer:
1
2
3
Click the Tray Widget. (See
.)
Click Tray Out, and remove the Utility Plate or Cell Plate.
To insert the tray and maintain the Target Temperature, click Tray In.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
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2
Use the Probes Widget to load/unload a Sensor Cartridge or raise/lower probes. To display options and select the appropriate action, click the
Probes
.)
Figure 41 Probes control widget
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 39
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During an assay, the Status Indicator light on the top of the XFe Analyzer will change color if a task requires user interaction or if an error has occurred, such as:
• To load a Sensor Cartridge or Cell Plate
• To remove a used Sensor Cartridge and/or Cell Plate
• To accept or cancel an assay if one or more wells did not calibrate properly after Calibration
• Any errors that can occur during the run, such as barcode read errors for the Sensor Cartridge, Cell Plate, or a protocol error.
The XFe Analyzer reads and records the Cell Plate and Sensor Cartridge barcodes before beginning an assay. A Barcode Read error will be displayed on
the rare occasion the barcode cannot be read. (See Figure 42 .) Contact Agilent
Seahorse Technical Support to assist with resolving this error and to start the assay.
40
Figure 42 Barcode read failure
For any Sensor Cartridge barcode read errors, Wave Controller will display the message shown in
and three corrective actions:
•
Open Tray: Eject the Sensor Cartridge to inspect barcode quality or to reverse the Sensor Cartridge.
•
Manual: Manually input the Sensor Cartridge barcode information. Contact
Agilent Seahorse Technical Support for this step.
•
Cancel Assay: Cancel the assay.
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1
2
To display the Cartridge Barcode window, click Manual. (See
.)
Call the appropriate regional Agilent Seahorse Technical Support telephone number to assist with entering the Sensor Cartridge barcode info for each
field on the form. (See Figure 43 .)
Figure 43 Cartridge barcode manual entry form
Figure 44 Cell Plate Barcode Read Failure
For any Cell Plate barcode read errors, Wave Controller will display the message shown in
and two corrective actions:
•
Manual: Manually input the Cell Plate barcode info.
•
Cancel Assay: Cancel the assay.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 41
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Figure 45 Cell Plate Manual Barcode Entry
1
2
3
4
5
Click the Tray Widget. (See
To eject the Cell Plate, click
Open Tray.
The Cell Plate barcode is located on the side of the plate. Write down the barcode information.
Click Close Tray.
On the Cell Plate Manual Barcode Entry window, enter the Cell Plate barcode, and click
42 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Agilent Seahorse XFe96 and XFe24 Analyzers For use with Wave Desktop
Wave 2.4 User Guide
Analysis View #1 - Quick View 45
Analysis View #2 - Overview 46
Analysis View #3 - OCR vs. ECAR 47
Introduction to Acidification Data 52
Data Analysis Using the XF Glycolytic Rate Assay Report Generator 57
Baseline to a Rate Measurement (%) 60
Baseline to a Control Group (%) 61
Standard Deviation and Standard Error of the Mean 61
Rate Overlay: OCR, ECAR, PER, PPR, O2, or pH 62
Modifying Assay Result Files 67
The primary purpose of Wave Desktop software is for data analysis of result files generated by Agilent Seahorse XF, Agilent Seahorse XFe, and Agilent
Seahorse XFp Analyzers. Wave Controller software for XFe Analyzers enables the same analysis features (for XFe data only), but analysis is strongly discouraged on the XFe Controller.
43
3
Analyzing Assay Results
Assay result files (*.asyr) are generated by XFe and XFp Analyzers only, and contain all data acquired during an assay. Examples of what type of data is acquired and stored in assay result files include:
• Rate data (OCR, ECAR, PER, and PPR)
• Level data (mmHg or mpH)
• Calibration results
• Consumable information from the barcodes on the cell plate and sensor cartridge used in the assay
In addition, any details added to the assay template file about the experiment, such as groups, compound, pretreatments, is migrated and saved in the assay result file. Most of this information can be edited using the
Modify function. For more information, see
“Modifying Assay Result Files" on page 67 for more
information. After completing an XF assay, save the result file to a USB flash drive (or network directory), and open the result file on your personal computer using Wave Desktop. The default analysis view for every new assay result file is called Quick View. Reopening a saved result file always displays the last-modified or viewed analysis view. Each analysis view can be added to an assay result file using the Add View button. The four analysis views in Wave software are:
• Quick View
• Overview
• OCR vs. ECAR
• Data
44 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
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Quick View is the default analysis view displayed when opening a new assay result file. Quick view simultaneously displays a kinetic graph of OCR vs Time,
ECAR vs Time, and a scatter plot of OCR vs. ECAR. (See
.)
Figure 46 Quick view
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Overview displays a kinetic graph of rate (OCR, ECAR, PER, or PPR) versus time.
The selected rate is displayed on the y-axis, and Time is fixed on the x-axis.
Group statistics (average rate and error) for each measurement can be displayed by checking the Details box in the Group List below the kinetic graph.
Overview is the most versatile analysis view in Wave software for most routine analysis functions. To display Overview, click Add View in the center of the ribbon menu, and select
Overview from the list of views. (See
46
N O T E
Figure 47 Overview view
Add multiple Overview analysis views repeating the process of Add View > Overview from the list of analysis views. This facilitates generating several customizable overview analysis views to display specific assay groups, or compare a control group versus experimental groups, groups treated with various compound concentrations or other variables measured in the assay.
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The
OCR vs. ECAR view displays the OCR on the y-axis and (by default) ECAR on the x-axis. To display OCR vs. ECAR, click Add View in the upper-left region of the screen, and select
OCR vs. ECAR from the drop-down menu. Use the rate drop-down menu to change the rate displayed on the x-axis to either PER or
PPR. OCR is always displayed on the y-axis, and cannot be changed.
Figure 48 OCR vs. ECAR view
The default graph title is
Energy Map. (See Figure 48 .) The corners of the
Energy Map have labels representing the relative bioenergetic state of the group(s) related to each other for the selected rate measurement. To modify the graph display, including the
Graph Title and the Quadrant Labels, click the
Settings cog. (See
.)
Figure 49 Graph display modification options
The Plate Map displays two rate measurements within each well (OCR vs.
ECAR, OCR vs. PER, or OCR vs. PPR) as indicated by the legend below the
Plate Map. (See
.)
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 47
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A kinetic graph is the most common way to display rate data from Agilent
Seahorse XF Analyzers. (See
Figure 50 .) The kinetic graph displays the rate
measurement on the y-axis, and the time on the x-axis. During an assay, data is acquired and plotted in real-time as a kinetic graph. Kinetic graphs are also found in the Quick View and Overview analysis views.
Figure 50 Kinetic graph
Another common way to express result data is a scatter plot of OCR vs. ECAR, where OCR is plotted on the y-axis, and ECAR plotted on the x-axis. (See
.) Data points are displayed on the OCR vs. ECAR scatter plot for a single rate measurement for each group in the assay. The OCR vs. ECAR graph is only available for analysis, not while running an XF assay.
Figure 51 Scatter plot
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The Plate Map displays rate data for the selected rate measurement of each assay well. (See
Figure 52 .) Plate Maps are always displayed in the upper right
corner of Wave while running an assay, and on the Quick View, Overview, OCR vs. ECAR analysis views. The Quick View has a button to display the Plate Map, which is hidden by default. The default rate measurement displayed on the
Plate Map is rate measurement 1.
Figure 52 Plate map
To change the measurement displayed in the Plate Map, use the Measurement
drop-down above the kinetic graph or scatter plot. (See Figure 53 .) The Rate
Highlight tool on the kinetic graph will move to the selected rate. Hide the Rate
Highlight tool by clicking
Options above the kinetic graph/scatter plot, and uncheck Show Rate Highlight.
Figure 53 Measurement drop-down
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The Bar Graph below the Plate Map displays the average rate for each group
(by default) for a selected measurement. (See
.) Use the Display drop-down menu to change the rate display from Group (average) to Well
(individual well) mode.
Figure 54 Bar graph
The Bar Chart graph is only available during analysis, not while running an assay. The Bar Chart view provides an alternate data display that can facilitate outlier identification, optimal FCCP concentration, optimal cell seeding density, or other trends that may not be obvious when viewing data as kinetic graph or scatter plot.
The Group List is the legend for the data plotted in the kinetic graph/scatter plot. (See
.) It provides several functions to modify the data plots:
• Hide groups from the data plot by double-clicking the name of the group. To unhide the data from the graph, double-click the hidden group again. When a group is hidden, the mean and standard deviation of the group will be:
Mean: 0.00 and Standard Deviation: 0:00.
• Display group statistics for the selected rate measurement by checking the
Details box in the upper-right corner of the Group List. Statistics are displayed as average and error for the selected rate measurement. Select a different rate measurement to display group statistics for that rate. Assay wells that have been turned OFF on the Plate Map are not included in the calculated group statistics.
Figure 55 Group list
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Use the Rate drop-down box to view the rate data options. (See
two rates measured by the Agilent Seahorse XF Analyzers are:
• Oxygen Consumption Rate: (OCR) (pmol/min)
• Extracellular Acidification Rate: (ECAR) (mpH/min)
Figure 56 Rate data drop-down
2
To view
Level data for O
2
and pH levels for each rate measurement during the assay, use the Y1: drop-down. (See
.) The O
2
level data is displayed as oxygen tension in units of mmHg. As the biological material in the well consumes oxygen during a measurement, the oxygen tension will decrease.
This decrease in oxygen tension during a used to calculate the rate of oxygen consumption (OCR). The pH level data displays the changes in pH for each rate measurement, and is used to calculate ECAR. Level data can be displayed while running an assay, and on the Overview and Data analysis views.
Figure 57 Y1 Drop-down
Figure 58 Oxygen Consumption Rate (OCR) data in Rate mode (left).
Oxygen tension (O
2
) data displayed as mmHg in Level mode (right).
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The Agilent Seahorse XF Analyzer sensor probes measure changes in the concentration of free protons in the microchamber in real-time; this data is referred to as Extracellular Acidification Rate (ECAR). ECAR can be transformed into a number that reflects the number of protons extruded over
time, Proton Production Rate (PPR), and Proton Efflux Rate (PER). Table 2
shows the types of acidification data.
Table 2
Types of acidification data
Type of acidification data:
Detects acidification from all sources?
Changes with media formulation?
Accounts for buffer factor of the system?
How buffering is accounted for?
Displayed while running assay?
ECAR
Yes
Yes
No
None
No
PER
Yes
No
Yes
Buffer factor
No
PPR
Yes
No
No
Buffer capacity
No
52 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
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PER is a new rate measurement calculated in Wave software. PER delivers a quantitative measure of extracellular acidification and is only available in post-run assay results. It is not displayed while running an assay. ECAR is the default reported acidification data in Wave software. The three rate options displayed in the
Rate Measurement drop-down menu are: OCR, ECAR, and PER
(previously PPR was the default). To change the reported acidification data option to PPR, use the
Options menu under Wave Home. PER is calculated using the following equation:
PER (pmol H+/min) = ECAR (mpH/min) × B.F. (mmol/L/pH) × Geometric Volume (µL) × Kvol
Table 3
Wave volume variables
Variable
Extracellular Acidification
Rate (ECAR)
Buffer Factor (BF)
Geometric volume
Unit
mpH/min mmol/L/pH
µL
Volume scaling factor (Kvol) No units
Description
Rate data measured during an assay, reported as the rate of change in mpH in an assay well.
Measure of the in situ buffer capacity obtained in
XF instruments, and accounts for both medium buffer capacity and XF assay conditions. For standard XF Glycolytic Rate Assay media, this value is prepopulated.
Physical (geometric) volume of the measurement microchamber.
Volume scaling factor used to account for total proton production in the measurement chamber.
Wave software calculates and reports total PER on the Overview, OCR vs.
ECAR, and Data analysis views for the following XF Analyzers: XFe96, XFe24,
XFp, and XF96. Use the
Rate Measurement drop-down menu to select PER as the rate to display on the kinetic graph (Overview analysis view), or scatter plot
(OCR vs. ECAR analysis view).
Wave automatically displays PER in two scenarios:
• When the Buffer Factor is automatically read from the sensor cartridge
• The assay media selected is the XF Glycolytic Rate Assay
The equation above demonstrates how PER is calculated in Wave software - the only user-changeable variable in the equation is Buffer Factor. A default Buffer
Factor is automatically used for the PER calculation. If you are using a custom
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 53
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assay medium, then Buffer Factor must be determined manually. Refer to the
Agilent Seahorse XF Buffer Factor Protocol (available in the XF Glycolytic
Rate Assay Report Generator download folder) for guidance.
Older result files in which the Buffer Factor is not included will show zero for
PER. PER will not be displayed:
• For XF24 and XF24-3 sensor cartridges
• For Spheroid and Islet plate types
• For assay template/assay result files that do not have the Glycolytic Rate
Assay Medium (DMEM-based) or (RPMI-based) selected, OR if a custom
Buffer Factor value is not entered.
• When background well Buffer Factor values have not been configured.
Figure 59 Assay Media option
3
4
1
2
Open the assay result file in Wave software.
Click
Modify.
Click
.)
To select the assay medium, use the
Media catalog drop-down menu.
(See
.)
• Glycolytic Rate Assay Medium (DMEM-based)
• Glycolytic Rate Assay Medium (RPMI-based)
54
Figure 60 Assay medium media menu
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3
6
7
5
To configure Background Well Buffer Factor, click Assay Media. (See
Click
Configure.
Under the column header
GRA Media, check the boxes next to each background well, and click
Figure 61 Configure background buffer factor
8
9
Assign the Assay Media to each group in the group list. First click
Collapse/Expand All to display the Group Definitions for each group.
Use the
Media drop-down menu to select the newly-added Glycolytic Rate
Assay Medium (DMEM-based) or (RPMI-based) for each group.
Figure 62 Media drop-down menu
10
When finished, click Apply.
11
The kinetic graph and scatter plot will display PER data when selected using the
Rate drop-down menu.
12
When finished with analysis, save the changes made to the result file.
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Prior to running the assay, calculate the Buffer Factor of the custom assay medium. Follow the steps in the Agilent Seahorse XF Buffer Factor Protocol
(available in the XF Glycolytic Rate Assay Report Generator download folder) for guidance.
1
2
Open the assay result file in
Wave software.
Click
Modify.
3
4
Click
Add next to Assay Media.
In the Edit Assay Medium window, enter the
Buffer Factor in the field provided.
5
6
7
Next, click Assay Media again to configure Background Well Buffer Factor.
Click Configure.
Under Buffer Factor (mmol/pH), type in the same Buffer Factor value and click
Save. (See
.)
56
Figure 63
8
9
Assign the Assay Media to each group in the group list. First click
Collapse/Expand All to display the Group Definitions for each group.
To select the appropriate medium for each group, use the
Media drop-down menu.
10
When finished, click Apply.
11
The kinetic graph and scatter plot will display PER data when selected using the
Rate drop-down menu.
12
When finished with analysis, save the changes made to the result file.
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Wave software calculates and reports total PER. If you performed the
XF Glycolytic Rate Assay, you must export data to the XF Glycolytic Rate
Assay Report Generator to calculate glycoPER, the rate of glycolysis-specific
proton efflux. (See Figure 64 .)
Figure 64
3
4
1
2
Open the assay result file in Wave software.
Click
Export.
Select the
XF Glycolytic Rate Assay Report Generator from the list.
Modify the file name or save location if necessary, then click
Save.
PER data is exported to both Microsoft Excel and GraphPad Prism file exports.
(See
Figure 65 .) For manual PER data analysis or graphing purposes, export
data from Wave to either file types using the Export function located in the top-level ribbon.
GraphPad Prism
Figure 65 Exported data examples
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
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PPR is calculated in Wave software using the following equation:
PPR (pmol H+/min) = ECAR (mpH/min) × Buffer Capacity (mol/L/pH) × Geometric Volume (L)
The default Acidification Data displayed in Wave software is PER, PPR is not available for selection in the Rate Measurement Drop-Down Menu until it is enabled in the Wave General Options:
1
2
3
Open
Wave software.
Click
Options.
Under
General Settings > Acidification Data click the radio button next to
Report PPR; Use Buffer Capacity (mol/L/pH).
4
5
Click Save.
Close/reopen Wave software for the changes to take effect.
Wave software reports PPR data on the Overview, OCR vs. ECAR, and Data analysis views when enabled, and is calculated using ECAR data and Buffer
Capacity of the assay medium used.
Buffer Capacity is required for calculating PPR from acquired ECAR data.
Buffer Capacity changes based on the constituents of the medium. Individual constituents have different abilities to buffer the medium from pH changes.
For accurate PPR data, the Buffer Capacity of each medium used must be measured and entered on Modify > Group Definitions tab in an assay result file.
1
2
3
Open an assay result file.
Click
Modify > Group Definitions.
Double-click
Assay Media (if an assay medium is not specified, use the Add button to add an assay medium).
58 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
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3
4
Click the assay medium and enter the
Buffer Capacity value. (See Figure 66 .)
If no Buffer Capacity is entered, the default value of 0.00078 is used. If more than one assay medium is used, repeat the same steps for each additional media.
Figure 66 Buffer Capacity value
1
2
3
Double-click Assay Media below Group Definitions.
Click Configure.
In the Configure Background Buffer Capacity window, type in the
Buffer
Capacity value for each background well listed. (See
assay medium is used, enter the same Buffer Capacity value. If more than one assay medium is used, enter the
Buffer Capacity of the assay medium for the appropriate background well.
Figure 67 Configure Background Buffer Capacity window
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The Baseline function in Wave software enables rate data to be expressed as a percent of a selected rate measurement, or as a percent of a control group. Use the
Baseline drop-down menu located on the Overview and OCR vs. ECAR analysis views (only). Select a Rate measurement to express kinetic data as a
percent of that rate measurement. (See Figure 68 .)
Raw OCR kinetic data expressed as (pmol/min)
OCR kinetic data expressed as a percent of rate measurement 3 (%)
Figure 68 Baseline to Rate measurement examples
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A new baseline feature allows rate data to be expressed as a percent of a
Control Group. The example shown in Figure 69 shows the appropriate use of
this feature. A Control group (blue line) with only media injections is selected as the baseline control group to baseline the two assay groups Glucose
Dependency (orange line) and Glucose Capacity (green line). Figure 69 shows
how this feature can provide a better graphical representation of the effect of glucose inhibition on OCR, and facilitate data interpretation.
Raw OCR kinetic data for the XF Mito Fuel Flex Test expressed as (pmol/min)
OCR kinetic date for the XF Mito Fuel Flex Test expressed as a percent (%) of the selected Baseline group (Control group)
Figure 69 Baseline to Control group examples
Two types of error bars in Wave are Standard Deviation ( StdDev) and Standard
Error of the Mean (
SEM). Use the Error Format drop-down menu to change the error bar type displayed on the kinetic graph/scatter plot and the Group List.
(See
Figure 70 .) to hide error bars from the data graph, select
None. In Well mode, error bars are not displayed.
Figure 70 Error Format menu
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2
Compare rates or level of data acquired during an assay from on one kinetic graph for one or more groups. Select data to overlay with the Y1 data using the
Y2 drop-down menu above the kinetic graph. (See
.)
Figure 71 Rate overlay
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By default, all assay wells on the Plate Map are turned ON when opening a new assay result file. Click an assay well to change the state of the well, excluded or included in the group statistics and graphed data for the select group. Assay wells that are turned off on the Plate Map have a grey background. (See
.) Assay wells that are included in the graphs and group statistics
have a white background. (See Figure 72 .) Assay wells must be manually
turned off from each analysis view. Turning off an assay well on one analysis view does not apply to other views.
Two assay wells in this group are turned OFF. These assay wells are excluded from the graph and group calculations.
All assay wells in this group are turned on and included in the graph and group calculations.
Figure 72 Assay well states
Column and row headers can be used to exclude entire vertical or horizontal sections of a Plate Map. Click the numerical column header or letter header for a row to turn the assay wells in that column or row OFF.
Click the small triangle in the upper-left corner of the Plate Map to turn off all
assay wells on the Plate Map. (See Figure 73 .) Charts on this analysis view will
not display any groups since all assay wells are turned off.
Figure 73 Plate Map toggles
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Rate data is graphically displayed in Group mode on all analysis views by default. Group mode displays the average rate value based on the assay wells in each group for each measurement. Group mode displays a single kinetic trace for each group on the Overview analysis view, and a single data point per group for each rate measurement on the OCR vs. ECAR view. In Group mode, error within each group is displayed as Standard Deviation or Standard Error of the Mean. Switch to
Well mode to display individual kinetic traces or data points for each assay well in each group. Error bars are not displayed in Well mode.
Group mode
Well mode
Figure 74 Example of an OCR kinetic graph in Group and Well mode
64 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
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View areas on the different analysis views can be adjusted in two ways: using
the gray borders (red arrows in Figure 75 ) and the << and >> arrows (outlined
in red in
) to hide entire regions of the view (Overview pictured in
). Stretch or reduce the size of the kinetic graph using the border between the kinetic graph and Plate Map. Hide the Group List to increase the vertical size of the kinetic graph using the border between the kinetic graph and Group List. Hide the Bar Chart using the border between the Plate Map and Bar Chart.
Figure 75 View areas
The Zoom, Pan, and Restore buttons are displayed above the kinetic graph on the Overview and the scatter plot on the OCR vs. ECAR view.
Zoom Right-click and hold to select an area on the kinetic graph or scatter plot to magnify the selected area.
Pan After zooming into a section of a graph, use the
Pan button to move around the graph while keeping the selected zoomed display aspect ratio.
Restore Return the graph to full view.
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To show the graph display formatting options menu, click the cog next to the restore button.
66
Figure 76 Graph display options
•
Minimum and Maximum: Set the minimum and maximum bounds for the Y1,
Y2, and X-axes.
•
Interval: Set the major units for the Y1, Y2, and X-axes.
•
Thickness: Add and adjust thickness of Y1, Y2, and X-axes gridlines. Adjust values in 0.50 intervals. Default value is '0.00' (no gridlines displayed).
•
Show Error Bars: Toggle error bars ON/OFF.
•
Show Zero Line: Display a horizontal reference line at the zero when the y-axis is negative.
•
Point-to-Point: Display point-to-point rates for the selected axis.
•
Show Rate Highlight: Toggle ON/OFF display of the vertical blue rate highlight bar on the kinetic graph (indicates the selected rate measurement that corresponds to values displayed on Plate Map).
•
Show Injections Marker: Toggle the vertical injection lines on the kinetic graph ON/OFF.
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Modify enables you to add or edit details of an assay result file. Modify is most-frequently used to add important experiment notes or add additional
Group Definition details (group names, compound injections, pretreatment details, and reassign assay wells to an outliers group or unassign assay wells from the Plate Map entirely). To display the Group Definitions, Plate Map,
Injection Names, General Information as tabs, click Modify (in the Functions ribbon). The Group Definitions is always displayed by default.
The views in Modify mode look almost identical to the template designer views. When actively modifying an assay result file, Wave will indicate the program is in Modify mode in the upper-left corner of Wave (upper-left corner of Wave). To return to assay result data, click Apply (upper-right corner of
Wave) to apply modifications to the assay result file, or click
Cancel to cancel out of Modify mode and prevent any changes from taking effect.
N O T E
The Modify function enables textual changes only, and is for adding notes or updating details of an assay. Wave does not allow or enable access to edit rate data acquired during the assay.
To display the Injection Strategies, Pretreatments, Assay Media and Cell Type details entered in the assay template prior to running the assay, select
Group
Definitions. Modifications to the Group Definitions tab include:
• Renaming assay groups
• Reconfigure details for one or more groups
• Add additional group information that was left out of the initial assay template design, or
• Add Buffer Factor to calculate PER
To display and edit conditions of each assay group using the drop-down menus below the group name, select
Plate Map. This tab also enables reassigning group locations on the Plate Map. select a group, then select the appropriate assay wells on the Plate Map.
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To edit the name of each compound injection during the assay, select
Injection
Names. The specified injection names from the Instrument Protocol tab are displayed above each injection line on a kinetic graph. The default name of each injection is: 'Injection 1', 'Injection 2', Injection 3', and 'Injection 4'. Type an Injection Name for each compound injection, and click
Apply to display the new name above the corresponding injection line.
To edit details of an assay result file, such as: Project Name, PI, Well Volume, or
Port Volumes, select General Information.
In the context of a Agilent Seahorse XF assay, normalization is the process of recalculating rate data to account for variations in a relevant cell parameter
(such as cell count or protein concentration) that can cause well-to-well variability. The Normalization function in Wave provides a simple method to apply normalization data to the measured rate data. To use the normalization function, an independent assessment of the assay wells for cell number, DNA content, protein concentration or similar parameter is required. (See
To normalize data in Wave, three components are used:
• Normalization Values (required): The numeric data generated from the independent assessment of the well (cell count, protein concentration, DNA content, and so forth).
• Normalization Unit (required): This alphanumeric field describes the units to which the data are to be normalized. It comprises the desired value to which the data will be normalized (see Normalization Scale Factor) plus the unit of measure of your normalization values (such as “cells”, “mg”,
“ng”, and so forth).
• Normalization Scale Factor: This number determines what value the rate data will be normalized to. Default is 1 and adjustment is optional.
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Figure 77 Normalization mode
To display the Edit Normalization Mode, click
Normalize (in the Functions ribbon). The Normalization Values table is where to copy/paste normalization data in Wave, two ways to add normalization data are:
4
5
2
3
6
1
Open the independent normalization data file, the data must be in the same grid format as the Normalization Values table.
Select the normalization data and copy to the clipboard [Ctrl+C].
Open a Agilent Seahorse assay result file in Wave Desktop.
Click
Normalize.
To insert the copied data the Normalization Table, click
Select All > Paste.
Click
Apply.
1
2
Open a Agilent Seahorse assay result file in Wave Desktop.
Click Normalize.
Type each value into the Normalization grid manually.
3
4
Click Apply.
Background wells should be left blank on the Normalization Table.
N O T E
Before applying normalization values, Wave requires a Normalization Unit.
The Normalization Unit is displayed on the Y1 axis title on the kinetic graph when normalization is applied and checked ON.
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N O T E
For example, type µg Protein into the Normalization Unit field, and the Y1 axis label for OCR will be displayed on the kinetic graph as: (pmol/min/µg Protein).
When using the Normalization Scale Factor, the scale factor value must also be entered to the Normalization Unit field.
The Normalization Scale Factor allows you to scale normalized data to a specific factor, such as cell number. It is often preferable to normalize to an average or rounded number.
Enter a scale factor value to recalculate and multiply normalized rate data based on the equation shown below:
Normalized Rate (well)=[Rate (well)/(Normalization Value)]*Normalization Scale Factor
For example, if assay wells contain values of 12052, 12503, and 12757 cells per well, a user might enter
10000
as the normalization scale factor.
Be sure to enter the same value in the Normalization Unit field to display the factor correctly on the y-axis. For example, if using cell count and the scale factor is 10000, enter
10000 cells as the Normalization Unit.
After applying changes made to the Normalize function, the Normalization checkbox will become active on all analysis views. Toggle the checkbox
ON/OFF to change the data display between normalized and nonnormalized rates on the kinetic graph, scatter plot, and Plate Map. All data graphs will display the Normalization Unit entered. (See
.)
70
Figure 78 Normalized data
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Assay result data can be exported from Wave for additional analysis, graphing, or other purposes. Use the Export button to display the available export options from any analysis view, or right-click a graph/Plate Map to display the menu options for exporting data. To export raw data, select Microsoft Excel or
GraphPad Prism options from the list. Create a customizable summary report by exporting data to an XF Report Generator. Select the XF Report Generator that matches the Agilent Seahorse assay type.
Export assay result data to Microsoft Excel from Wave for additional data analysis or graphing purposes.
1
2
3
Click
Export (in the Functions ribbon).
Click
Microsoft Excel.
Choose a save location, and click
Save.
Using the export function described above, there will be between 7–13 tabs in the exported Excel file depending on the data configured in Wave:
• Raw data: Exporting raw result data (not normalized, baseline OFF) will have seven tabs of data. (See
.)
Rate (Plates) Tab: Raw OCR rates for rate measurement 1 (from an
Agilent Seahorse XFp Analyzer).
Figure 79 Raw data
a b
Assay configuration: Information from the assay result file including the Group Layout (Plate Map), Cartridge/Cell Plate Barcode and other information, Instrument Protocol, and normalization data and normalization unit.
Rate: Raw OCR, ECAR, and PER or PPR data in a column format organized by measurement.
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Rate (Columns): Raw OCR, ECAR, and PER or PPR data in a column format organized by group. Similar output format to the GraphPad Prism export.
Rate (Plates): Raw OCR, ECAR, and PER or PPR data organized by measurement displayed as a Plate Map.
f e g
Raw: Raw assay well data for temperature, emission values and reference values. Often used for troubleshooting by Agilent Seahorse
Technical Support.
Calibration: Raw calibration data for both the O2 and pH channels.
Operation log: List of each protocol command executed by the analyzer, timing of the command, and outcome (success/fail).
• Normalized data: Exporting result data that has been normalized (baseline
OFF) will have three additional data tabs in the Excel export.
(See
.)
Normalized Rate (Plates) Tab:
Normalized OCR rates for rate measurement 1 (from an Agilent
Seahorse XFp Analyzer).
Figure 80
Normalized data
a b c
Normalized rate: Normalized OCR, ECAR, and PER or PPR data in a column format organized by measurement.
Normalized rate (Columns): Normalized OCR, ECAR, and PER or PPR data in a column format organized by group. Similar output format to the
GraphPad Prism export.
Normalized rate (Plates): Normalized OCR, ECAR, and PER or PPR data organized by rate, displayed as a Plate Map.
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• Baselined data: Exporting result data with baseline ON (recalculated as a percent of a rate measurement or of a control group) will have three
additional data tabs in the Excel export. (See Figure 81 .)
Baselined Rate (Plates) Tab:
Baseline OCR rates for rate measurement 1 displayed as a percentage (from an Agilent
Seahorse XFp Analyzer).
Figure 81 Baseline data
a b c
Baselined rate: Baselined (%) OCR, ECAR, and PER or PPR data in a column format organized by measurement.
Baselined rate (Columns): Baselined (%) OCR, ECAR, and PER or PPR data in a column format organized by group. Similar output format to the
GraphPad Prism export.
Baselined rate (Plates): Baselined (%) OCR, ECAR, and PER or PPR data organized by rate, displayed as a Plate Map.
The legend on each tab indicates the assay wells that have been excluded from
calculations in Wave (turned OFF or unassigned). (See Figure 82 .) Assay well
B in the images below was turned OFF in Wave before exporting to Excel.
Figure 82 Excel export legend
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If you wish to only export select data that is displayed on a kinetic graph, scatter plot, or Plate Map to Microsoft Excel.
1
Right-click anywhere on a chart or
Plate Map to display an option menu and select
Export Graph Data to Excel. (See Figure 83 .)
N O T E
Figure 83 Option menu
2
3
Browse to the desired save location, type in the file name for the export.
Click
Save.
The data values exported to Microsoft Excel will be in the exact same format as they are displayed on the chart or Plate Map.
This Microsoft Excel file export is not compatible with the XF Report Generators. Use the
Export button in Wave software to export data directly to the XF Report Generators from any analysis view.
Export assay result data to GraphPad Prism for additional data or statistical analysis, or graphing purposes.
N O T E
1
2
Click
Export (on any analysis view).
Select
GraphPad Prism.
3
Choose a save location, and click
Save.
Prism export compatibility has been validated using GraphPad Prism versions 6 and 7.
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Wave exports rate data (OCR, ECAR, PER or PPR) to GraphPad as:
• Raw rate data: always exported
• Normalized rate data: Normalization data must be applied to the assay result file in Wave before export
• Baseline rate data: Rate data as a percent of the selected baseline measurement or control group
Sample data output in GraphPad Prism from an Agilent Seahorse XFp
Analyzer with two assay groups,
Disease Group and Healthy Group.
(See
Figure 84 Sample data output
• Column-format Data Display: Time is a fixed column next to the assay groups, and will scroll horizontally when there are more assay groups.
• Assay Wells and Columns: The number of columns with rate data (below each group header) reflects the number of assay wells in each group.
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• Excluded Assay Wells: Assay wells that have been excluded in Wave will appear as an empty column in Prism. Column A:Y3 below Group A (called
Disease Group) in Figure 84 on page 75 shows rate data that has been
exported for two wells in the Disease Group. Refer to Project Info or the assay result file in Wave for excluded assay wells in the exported Prism file.
• Rows and Rate Measurements: The number of rows reflects the number of
rate measurements captured during the assay. In Figure 84 on page 75,
there are a total of 16 measurements performed during the assay (16 rows).
Every Data Table in the GraphPad Prism file export also contains a corresponding kinetic graph. The graph in
represents the data set on the previous page from the Agilent Seahorse XFp Analyzer. The Graphs folder displays the available kinetic graphs based on the type of data exported from
Wave, that is, raw data, normalized data, or data as a percent of the baseline.
Assay wells or groups that are turned OFF in Wave prior to export will be excluded from the file export.
Figure 85 Kinetic graph
Information about the experiment and assay properties is displayed in Project info 1 (in the Info folder) including the date the assay was performed, the
assay result file name, and normalization unit. (See Figure 86 .) The Notes
display on the Project Info view shows the group layout, assay wells in each group, and indicates excluded assay wells using brackets (example: [A2]).
76
Figure 86 Project info view
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Agilent Seahorse XF Report Generators are Microsoft Excel macro files that automatically calculate the parameters of the selected Agilent Seahorse XF assay, and present the data in a one-page, customizable Summary Report.
Wave enables a simple, one-click direct export of result data to the Report
Generators. Any modifications in Wave will be incorporated in the exported
Report Generator file, including excluded outlier assay wells and normalizing data.
The five Agilent Seahorse Report Generators embedded in Wave are:
• XF Glycolysis Stress Test Report Generator
• XF Glycolytic Rate Assay Report Generator
• XF Cell Energy Phenotype Report Generator
• XF Cell Mito Stress Test Report Generator
• XF Mito Fuel Flex Test Report Generator
N O T E
Figure 87 Embedded report generators
The Agilent Seahorse XF Report Generators are validated for compatible with MS Excel
2010, 2013, and 2016 on Windows PCs and MS Excel for Mac 2011 and 2016 for Apple PCs.
For more information on a specific XF Report Generator, refer to the appropriate Report
Generator User Guide on the Agilent website.
1
2
Open assay result file and modify data as necessary.
Click
Export.
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N O T E
3
Select the Agilent Seahorse XF Report Generator that applies to the result data.
4
5
The Report Generator file name matches the assay result file name by default. Enter a new file name if desired, select a save location, and click
Save to save the Report Generator file.
Open the
Report Generator file, and select groups to display. The Select
Groups dialogue box is displayed automatically the first time data is exported to the XF Report Generator.
6
To save the Summary Report, click
Save.
When exporting an assay result file that has data from two different assays on the same
Cell Plate (such as a Agilent Seahorse XF Cell Mito Stress Test and Glycolysis Stress Test), turn OFF groups that do not apply to the Report Generator you are exporting to first. Repeat the same process for the other assay groups and Report Generator.
Before using a XF Report Generator, macros must be enabled. Macros are disabled with notification in the Microsoft Excel
Trust Center by default. Opening a newly-exported Report Generator will display a security warning to enable the macro. To use the XF Report
Generator, click
Enable Content. This must be enabled for subsequent XF Report
Generator file exports.
To always enable macros and not be prompted to
Enable Content each time a
Report Generator file is created, enable all macros in the Microsoft Trust
Center.
1
2
Open
Microsoft Excel.
Click
File>Options.
3
4
5
Click
Trust Center>Trust Center Settings.
Click
Macro Settings.
Select
Enable all macros, and click OK.
Normalized rate data in Wave will be exported and used for parameter calculations in the Report Generator.
Normalized data is displayed by default after selecting groups to display. To toggle the rate data display between normalized and raw data, use the
Normalize button on the Summary Printout page. The Normalize tab in the
Report Generator will display the normalization values, unit, and scale factor as exported from Wave. (See
Figure 88 on page 79.) For data integrity
purposes, when normalized data is exported to a Report Generator from Wave, the Normalize tab in the Report Generator is locked for editing.
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Figure 88 Normalize tab view
The Normalize tab in the Report Generators provides a step-by-step explanation on how to edit the normalization values imported to the Report Generator.
Figure 89 Instructions to edit the normalization values
Assay wells that are turned off in
Wave will not be exported or included in parameter calculations for each group in the Report Generators. This also applies to entire groups in an assay result file. Groups turned off in Wave will not be exported to selected Report
Generators. The Measures Sheet displays the group names, Plate Map layout, and any assay wells that have been excluded in the group calculations in the
Report Generator. (See
.)
Figure 90 Measures sheet
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The Summary view is added to every assay result file by default, and cannot be closed or deleted. The Summary is a place to add notes or other valuable information about the assay. You may also append graphs, charts, Plate Maps from other views, or add details of the Instrument Protocol or
Groups/Conditions used in the assay. The Summary is the only location in
Wave that enables export to Microsoft Word, Adobe PDF, HTML, or Rich Text
.)
Figure 91 Summary view
Use the ribbon toolbar to add, edit, and format edit the content added to the
Summary, such as:
• Cut, copy, and paste to the clipboard.
• Change the font type, size, text, highlight color, and style.
• Subscript or superscript a character or characters.
• Add bullet points or numbers.
• Insert a table, picture or page break.
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Append content from any analysis view directly to the Summary in three ways:
• Append to Summary: Click Summary (in the Functions ribbon) on the Quick
View, Overview, OCR vs. ECAR views to send all data on the current analysis view to the Summary.
• Right-click > Send to Summary: Right-click a kinetic graph, scatter plot, or
Plate Map and click
Append to Summary from the drop-down list. To view appended content, click the Summary view.
• Append Image (Summary view): General Information: The General
Information button appends an image to the Summary view containing information about the assay, including project and plate information.
•
Protocol Summary: The Protocol Summary appends the Instrument
Protocol (Mix, Wait, and Measure steps) as well as the time and number of cycles for each measurement to the Summary view.
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•
Group Summary: The Group Summary button appends an image of the
Plate Map and a table of the Group Definitions to the Summary view. The
Plate Map is shown below.
•
Port Summary: The Port Summary button appends an image to the
Summary view of the contents injected from each compound port in the assay.
82
To print the Summary view, click the Print button on the Summary toolbar, select the printer to use, and click
Print.
Export the contents of the Summary view to a Microsoft Word Document, PDF file, HTML file, or Rich Text Format file. Click the appropriate icon from the
Export section of the Summary toolbar, browse to the desired file location to save and click Save.
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The Data view contains every piece of data associated with assay result file, such as:
• Group Data: OCR, ECAR, PER, or PPR rate data and error values organized by group, and ordered by measurement.
• Rate: OCR, ECAR, PER or PPR individual assay well data. Error values are not displayed.
• Level Data: Individual assay well data displayed as Level data - O2 level
(in mmHg) and pH raw values.
• Raw: Raw data values obtained during the assay including LED values, temperature at each measurement, and internal LED reference values.
• Calibration: Calibration results for each assay well are displayed in a table format.
• Calibration View: Calibration results for each assay well are displayed in a
Plate Map format.
• Event Log: XF Analyzer serial number, consumable lot numbers used during assay, a command log for processes performed during assay, and whether they were successful or failed.
Export the data in a selected tab to Microsoft Excel using the right-click
Export function. The Calibration, Raw, and Event Log tabs are used primarily for internal purposes, including QC, validation processes, and Agilent Seahorse
Technical Support.
To display the Data view, click
Add View on the top-level ribbon and select Data from the drop-down menu. The Group Data tab is displayed by default when opening the Data view. (See
.)
Figure 92 Data view
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To sort the data by a specific column, click on the column header:
Measurement, Group Name, Time, Rate or error for OCR, ECAR, or PER/PPR.
Wave will automatically sort the data in ascending or descending order in the selected column.
To display the Average, Count, Maximum, Minimum, or Sum, or any combination of these values, click the Sigma sign to the right of the arrow, and check the applicable boxes. In the example shown below, all values are checked for OCR data - Wave will display the average, count, maximum value, minimum value, and sum for the OCR data.
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To group the data by field, drag and drop a column header to the gray bar above the table. In the example shown below, the Group Name heading was dragged to the gray bar. After grouping the data, expand or collapse the grouped by clicking on the plus/minus sign to the left of the group.
After expanding a group, select another column heading to define the groupings further. In the example below, the heading OCR was dragged to the grey bar after Group Name.
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Managing Agilent Seahorse Files
Manage the list of available templates for the Agilent Seahorse XFe and XFp
Analyzer on the Templates view,
Import, Export,
Duplicate, and Delete buttons.
Figure 93 Templates view
Import assay template files to Wave two ways:
• Double-click the assay template on a USB flash drive, network drive, or shared in an email, and Wave will automatically import the template to the
Templates view.
• Manual Import - Open
Wave > Templates view and click Import. Locate the template file, and click Open.
Single-click an assay template on the Templates view to:
• Duplicate: Create an identical copy of a template.
• Delete: Permanently delete an assay template file from Wave.
• Export: Export an assay template to:
•
A USB flash drive
•
A shared network directory for multiple user access
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To display details about an assay template without opening the template file, single-click the template to display
Template Details. (See Figure 94 .) The
Modified date reflects the most recent date the template was modified and changes were saved. Description, Project Name, Project Number, and Investigator are optional details and not required to save a template.
Figure 94 Template Details
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, displays recently opened assay result files, the corresponding file directory where each assay result file is located, and
Favorite Places. Opening a new assay result file displays the default analysis view, the Quick View. After modifying and saving an assay result file, Wave automatically displays the last modified analysis view the next time the assay result file is opened.
Figure 95 Results view
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Figure 96 Recent Assays, recent places, and favorite places
•
Recent Assays displays a sortable and searchable list of recently viewed and analyzed assay result files. (See
•
Favorites displays user-defined locations (local or network directories) where assay result files are frequently added to, or accessed from. Once configured, each local directory added as a Favorite Place will appear below
Recent Assays as a folder icon. (See Figure 96 .)
•
Recent displays the file location (for both local and network directories) of recently opened assay result files in the order of when each result file was opened. (See
.)
Click
Options, next to Browse in
Figure 95 on page 90, to add Favorite Places
(from local or network directory locations) or configure the number of
Recent
Folders and Recent Assay Files to display on the Results view. (See
Figure 97 Number options for results view
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N O T E
To manually open an assay template or assay result file, click
Browse. (See
The file types compatible with Wave 2.4 are:
• Assay result files (*.asyr)
• Assay templates (*.asyt)
• Agilent Seahorse XF data files (*.xfd)
Double-click to open an assay design file (*.asyd) created by earlier versions of Wave and
Save As an assay template file. It is not possible to create assay design files using
Wave 2.4. If you have *.asy files, these must be converted to *.asyr before opening in Wave.
Contact Agilent Seahorse Technical Support for help with this.
Assay result files are default-sorted by
Most Recent, which displays a list of the most-recently opened or modified assay result file first. Name sorts assay result files alphabetically based on the file name and
Instrument sorts by the type of Agilent Seahorse XF Analyzer used to generate the assay result file.
Figure 98 Sort by from the results view
Type specific keywords in the Search bar to filter and display assay result files whose file name contains matching keywords. Searching using the file directory or date is not supported.
Figure 99 Search from the results view
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The
Catalog provides a location to save frequently used Compounds,
Pretreatments, Media, and Cells. (See Figure 100
.) Save time when creating a new assay template or customizing a template file by inserting Catalog entries.
The Catalog is prepopulated with Agilent Seahorse reagents and compounds that are used in commercially available Agilent Seahorse assay kits.
Figure 100 Catalog
1
2
4
5
3
Open the
Catalog view.
Select a condition (
Compounds, Pretreatments, Media, and Cells). This example used Pretreatments.
At the bottom of the Catalog > Pretreatments view, type in a Name (required), and any additional fields to describe the
Pretreatment entry (optional).
After adding details, click
Add Pretreatment to save the entry.
When finished, click
Save in the lower-right corner of the Catalog view.
1
2
Open the Catalog view.
Click the red X at the end of the row to delete.
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User-customized Catalogs can be shared and uploaded to Wave on XFe
Analyzer or another user’s Wave Desktop.
1
2
3
4
Plug in the USB flash drive (if applicable).
Start
Wave, and open the Options view.
The
Catalog directory is found under the Directories header on the General
Options view.
Highlight the path and copy/paste into Windows Explorer to display the
Catalog folders: CellLine, Compound, Pretreatment, and XFeMedia.
5
6
Select each Catalog condition to share and copy/paste to the USB flash drive (or a network directory). Do NOT cut the Catalog entries.
Eject the USB flash drive (if applicable).
2
3
4
5
6
7
1
Insert the USB flash drive (or open the network directory) containing the saved Catalog entries.
Start
Wave, and open the Options view.
In
General Options, copy/paste the Catalog directory location in Windows
Explorer.
Close Wave.
In Windows Explorer, open each Catalog folder (from Computer #1), and copy/paste the contents into the appropriate
Catalog folder on
Computer #2.
Eject the USB flash drive (if applicable) and open Wave.
Open the Catalog view, and verify the transferred entries are displayed in the list for each condition.
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The three types of modifiable settings in the
Options view are: General,
Instrument, and Advanced. (See Figure 101 .)
Figure 101 Options view
General settings displays options to:
• View/change Agilent Seahorse file directories (save locations) for Catalog and Assay Templates files in Windows Explorer.
• Modify the reported acidification rates - PER (default) and PPR.
• Enable/disable the
What’s New pop-up (list of new features) upon startup of
Wave 2.4 software.
• Enable/disable automatic Wave software updates.
After making changes to
General options, click Save (lower-right corner) to save changes. To restore changes to the factory default setting, click the Default button.
Directories displays the default save location for Catalog entries and Template files. (See
.) The prepopulated Catalog conditions and assay template files installed with Wave are stored in this directory, as well as any new Catalog entries and assay template files. To change the location of either directory, click the [ …] button to browse to the preferred location. Wave software must be restarted for changes to take effect.
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The What’s New message box appears upon Wave startup, and displays the new features and functions available in version 2.4. This message box will always appear unless either the Don’t show this again box is checked, or it is disabled in the
Options view.
Instrument options displays default values (by analyzer type) for the
Instrument Protocol. (See Figure 102 .) To change the XF Analyzers displayed
in Wave Desktop, see the Wave Desktop 2.4 ReadMe PDF.
96
Figure 102 Instrument options
Other settings that can be modified include:
•
Cycles: Number of repeat Mix, Wait, and Measure cycles in the default protocol
•
Mix, Wait, and Measure times: Time to complete each command in the
Instrument Protocol
• Default
Port Volume (notation purposes only)
• Default
Well Volume (notation purposes only)
Leave the default Protocol Defaults untouched, and modify the Instrument
Protocol in the individual assay template file. To change the
Protocol Defaults:
1
2
Use the up/down arrows or type in the new values for
Mix, Wait, Measure, and Cycles.
Click Save at the bottom of the screen to save changes.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Managing Agilent Seahorse Files
4
The port and well volumes are unique to each Agilent Seahorse analyzer.
Modifying these values do not change the function of the analyzer or calculations, and are for record-keeping only. During an assay, the contents of the port will be injected. To change the
Port Volume or Well Volume:
1
2
Use the up/down arrows, or type the new value in the
Port Volume field or the Well Volume fields.
To save changes, click Save.
The Agilent Seahorse XFe Analyzer can automatically notify users when certain functions are complete or need user interaction, such as:
•
Calibration: The Calibration Plate must be replaced with the Cell Plate, and the assay must be started.
•
Assay Complete: Following completion of an assay, Wave Controller will automatically email the assay result file to the specified email addresses.
An active internet connection configured on Wave Controller for the Agilent
Seahorse XFe Analyzer is required for use of automatic email notification features. (See
.)
Figure 103 Automated Email features
2
3
4
1
5
Type an email address in the
Mail From field, and enter a password for this address in the Password field. This email address will be used to send all notifications and will be displayed in the
Mail From field after receiving a notification from the Agilent Seahorse XFe Analyzer.
Specify the SMTP Address and the access Port field.
Check Enable SSL if required by the local IT group.
Type the email address for a single recipient in the Recipient Email Address field.
Click
Add.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 97
4
Managing Agilent Seahorse Files
6
7
8
Repeat Steps 4 and 5 for each recipient.
When finished adding recipients, click Save.
To send a test notification email to all email recipients, click Test Settings. If the test email is not received, verify the correct network settings have been configured in Wave Controller.
Select the email address under
Email Recipients, and click Delete.
98 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
Managing Agilent Seahorse Files
4
To quickly and easily send system files for support and diagnostic purposes, the Help view displays software version information, Agilent Seahorse technical support contact information, and
.)
Figure 104 Help view
Use the Send System Files function to automatically compile Wave System Files into a compressed folder, create a blank email to attach the System Files, and send to Agilent Seahorse Technical Support. (See
.)
Figure 105 Send system files function
1
2
3
Open the Help view.
Click Send System Files.
Click Save to select a file location to save the compressed folder.
Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide 99
4
Managing Agilent Seahorse Files
3
4
1
2
Open the Help view.
Click Send. An email message will appear populated with the following information:
a b
Email Recipient - Agilent Seahorse Technical Support email address.
Subject - Email subject line.
c
Email Body Text - displays the default save location of the System Files on the local drive.
Locate the saved System Files compressed folder, and attach it to the email.
Click Send.
100 Agilent Seahorse XFe96 and XFe24 Analyzers Wave 2.4 User Guide
© Agilent Technologies, Inc.
Printed in USA, June 2017
For Research Use Only
Not for use in diagnostic procedures.
*S7894-10000*
S7894-10000
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How to set up multiple experimental conditions on a single plate in the Wave software?
Define unique groups by combining Injection Strategies, Pretreatments, and Assay Media in the Group Definitions step, then assign these groups to the Plate Map manually or by using the 'Distribute Groups' feature.
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