1
Report Generator
User Guide
Agilent Seahorse
XF Cell Mito Stress Test
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Table of Contents
Introduction .............................................................................................................................................................................................................. 3
Parameter Calculations ........................................................................................................................................................................................ 3
XF Report Generator Overview ............................................................................................................................................................................ 4
How to: ..................................................................................................................................................................................................................... 4
Configure Microsoft Excel to Enable Macros ................................................................................................................................................. 4
Analyze Data in the Report Generator ............................................................................................................................................................ 5
Select Groups and Display Results ................................................................................................................................................................. 5
Save a Summary Report .................................................................................................................................................................................... 6
Advanced Options .............................................................................................................................................................................................. 6
Acute Injections .................................................................................................................................................................................................. 6
Error Bar Calculations ........................................................................................................................................................................................ 7
Normalize Assay Results .................................................................................................................................................................................. 7
Exclude Assay Wells (outliers) or Groups from Report Generator Analysis ........................................................................................... 8
Frequently Asked Questions ................................................................................................................................................................................ 8
Feedback ................................................................................................................................................................................................................... 9
Glossary .................................................................................................................................................................................................................... 9
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Introduction
Agilent Seahorse XF data analysis and interpretation – accelerated. In a few simple steps, transform Agilent Seahorse XF Cell
Mito Stress Test assay result data into interpretable, compelling data tables and figures of mitochondrial function for each group
analyzed using the Agilent Seahorse XF Cell Mito Stress Test Report Generator. Easily copy figures or data tables from the
Report Generator to other software programs for additional graphing or statistical analysis and eliminate tedious, error-prone
steps in any XF data analysis workflow.
The Seahorse XF Cell Mito Stress Test Report Generator is a Microsoft Excel Macro that automatically calculates and reports
assay parameters of the Seahorse XF Cell Mito Stress Test (Table 1) as absolute Oxygen Consumption Rate (OCR) in pmol
O
2
/min and supports assay result data from all Agilent Seahorse XF Analyzers.
Parameter Calculations
Parameter calculations performed in the
Seahorse XF Cell Mito Stress Test Report
Generator are outlined in Table 1 below.
Each parameter value calculated
represents the average of individual well
calculations for each assay group on the
Plate Map. Error bars are calculated
based on the individual well calculations
for each parameter. See the Calculations
tab in the Report Generator for more
information and example calculations.
Table 1 | Seahorse XF Cell Mito Stress Test Parameter Equations
Parameter Value Equation
Non-mitochondrial Oxygen
Consumption
Minimum rate measurement after Rotenone/antimycin A injection
Basal Respiration
(Last rate measurement before first injection) – (Non-Mitochondrial Respiration
Rate)
Maximal Respiration
(Maximum rate measurement after FCCP injection) – (Non-Mitochondrial
Respiration)
H+ (Proton) Leak
(Minimum rate measurement after Oligomycin injection) – (Non-Mitochondrial
Respiration
ATP Production
(Last rate measurement before Oligomycin injection) – (Minimum rate
measurement after Oligomycin injection)
Spare Respiratory Capacity (Maximal Respiration) – (Basal Respiration)
Spare Respiratory Capacity as a % (Maximal Respiration) / (Basal Respiration) × 100
Acute Response
(Last rate measurement before oligomycin Injection) – (Last rate measurement
before acute injection)
Coupling Efficiency ATP Production Rate) / (Basal Respiration Rate) × 100
Figure 1 | Agilent Seahorse XF Cell Mito Stress Test assay parameters and kinetic profile.
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XF Report Generator Overview
The Seahorse XF Cell Mito Stress Test Report Generator displays result data and other assay-related information on 5-6 tabs.
For the optimal Report Generator data analysis experience, update to Wave 2.3
.
1. Summary Printout: One-page Summary Report of the imported Seahorse XF Cell Mito Stress Test result data presented as a
kinetic graph and bar charts for select assay parameters.
2. Bar Charts: Assay parameter data displayed as bar charts.
3. Normalize: Plate map of normalization values applied in the imported result file. This tab is only displayed for result files
that have been normalized in Wave 2.3 before Excel export. See the Wave User Guide
for more info.
Note: Assay result data that has not been normalized in Wave 2.3 will not be display the Normalize tab.
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4. Measures Sheet: Result data organized in tabular format for assay parameters and kinetic rate data for both OCR and
ECAR.
5. Assay Parameter per Well: Data table of assay parameters per well for each group in the imported file.
6. Sample Calculations: Example parameter calculations using sample data.
How To:
The following sections describe how to perform routine functions in the Report Generator:
• Analyze Data in the Report Generator
• Save a Summary Report
• Normalize Assay Results
• Exclude Outlier Wells
Configure Microsoft Excel to Enable Macros
The Seahorse XF Cell Mito Stress Test Report Generator is a Microsoft Excel Macro-Enabled Template and is compatible with
Microsoft
®
Excel™ versions 2010, 2013 and 2016 for Windows PCs, and Microsoft
®
Excel™ for Mac versions 2011 and 2015. In
order to use this Report Generator, Excel must be configured to allow macros to run. To enable macros once, double-click the Multi-
File_Seahorse XF Cell Mito Stress Test Report Generator.xltm file, then click Enable Editing and Enable Content (yellow information
bar) if prompted (Figure 2).
To always enable macros (recommended for the best experience using Report Generators):
1. Open Microsoft Excel.
2. Click File, then click Options.
3. Click Trust Center, then click Trust Center Settings.
4. Click Macro Settings.
5. Select Enable all macros.
Figure 2 | Enable macros using the Enable Editing button as seen on the yellow information bar. This needs to be performed once upon opening the Report
Generator for the first time after download.
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Analyze Data in the Report Generator:
*Recommended*
Direct-export from Wave 2.3:
The recommended method to analyze assay result data in the Report
Generator is using the direct export from Wave 2.3 (Figure 3):
1. Transfer the assay result file to a personal computer using a USB
flash drive or shared network directory.
2. Double-click to open the assay result file in Wave Desktop 2.3.
3. Click Export.
4. Select the Seahorse XF Cell Mito Stress Test Report Generator.
Optional: Modify the default file name and save location.
5. Click Save.
Import Excel file manually:
1. In Wave 2.3, click Export.
2. Select Microsoft Excel and click Save.
Optional: Modify the default file name and save location.
3. Download the Seahorse XF Cell Mito Stress Test Report Generators from the Agilent website.
4. Unzip the compressed folder and open the Report Generator file.
5. Click Load New Data File.
6. Locate the Microsoft Excel file (exported from Wave 2.3) and click Open.
Select Groups and Display Results
After exporting assay result data from Wave 2.3 to the
Report Generator (or manually importing Excel data), use
the Display Options dialogue window to select groups
from the assay to display and click Update Summary
(Figure 4). The Report Generator will automatically
calculate the parameters for each group selected and
display results on the Summary Printout tab.
Error Bar Type
Standard Deviation is selected as the default Error Bar
Type for ALL graphs. The Error Bar Type applies to all
graphs in the Report Generator.
Figure 4 | Group names configured in Wave shown on the Display Options window
when selecting groups in
the Report Generator.
Figure 3 | Wave 2.3 (Desktop & Controller) export
options.
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Save a Summary Report:
*Recommended*
After exporting from Wave 2.3:
When exporting data from Wave 2.3 to the Report Generator, Wave will automatically create a unique Summary Report file
(Microsoft Excel Macro-Enabled Worksheet) for each exported result file. Additional data files cannot be loaded into this Report
Generator file and must be created using the direct-export from Wave 2.3.
.
• Save/Save as: Click the Save icon (small floppy disc) to display the Save as function. Select a file location and enter a
custom file name if desired. The saved Summary Report can be re-opened to view the calculated parameters for the selected
groups, format/customize the appearance of graphs and figures or select new groups from the assay to run through the
Report Generator. The Report Generator default file type is a Microsoft Excel Macro-Enabled Template (*.xltm) – this file
cannot be overwritten.
• Save As – Excel Workbook: Use the Save As function to save the customized Summary Report as an Excel Workbook file
format (*.xlsx).
• Save As – PDF: Use the Save As function to save the customized Summary Report as a PDF file format (*.pdf).
Note: Saving the Report Generator as an Excel workbook or any other file type than the default file type (Excel Macro: *.xlsm) will render the Report Generator
macro inoperable – modifying the groups selected or importing additional assay result data is not supported as an Excel Workbook file type.
Advanced Options
Advanced Options (Figure 5) is accessed from the Display Options window when
selecting groups to display by clicking Advanced. Advanced Options displays the
Instrument Protocol, which is automatically imported into the Report Generator
from Wave or the Excel file. Instrument Protocol displays the number of injections
performed file and measurements before injection and after each injection.
Acute Injections
An injection that occurs in the Seahorse XF Cell Mito Stress Test assay following the baseline measurements, but before the
Oligomycin injection is called an Acute Injection. The default Seahorse assay template in Wave (Desktop or Controller) called
“Seahorse XF Cell Mito Stress Test (Acute Injection)” has the specific Instrument Protocol for acute injections and should be
used for every Seahorse XF Cell Mito Stress Test assay with an acute injection. For custom assays, this injection step must be
manually added to the Instrument Protocol in Wave (Desktop or Controller) prior to the assay.
• An acute injection must be injected before oligomycin from Port A on the cartridge.
• The Acute Injection checkbox is enabled automatically when a 4
th
injection is detected in the imported Excel file (Figure 5).
• Parameter calculations in Table 1 are shifted by the number of acute response measurements for assays with an Acute
Injection.
• Spare Respiratory Capacity in the presence of an acute injection is calculated using the equation:
(
Maximal Respiration
)
− (Last Rate Measurement before Oligo Injection)
Figure 5 | Instrument Protocol displayed in Advanced
Options
.
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Error Bar Calculations
Error Bar Type is a universal setting and applies to ALL graphs and charts in the Report Generator. Standard Deviation is the
default error bar type. To change the error bar type to Standard Error of the Mean (S.E.M.), click Edit Current Group Selection and
select Standard Error.
•
Error Bars are calculated from each replicate of the rate measurement used to determine the assay parameter (see Table 1).
•
Standard Deviation is calculated using the Microsoft Excel function.
•
S.E.M. is calculated using the equation:
(
)
�
(
)
Normalize Assay Results:
It is highly recommended to analyze rate data that has been normalized to a cellular or mitochondrial parameter compared to
non-normalized, raw rate data. Normalization data must be added to the Normalize view in Wave 2.3 (Figure 6a) before export –
normalized rate data will be used for parameter calculations and displayed by default in all kinetic graphs and bar charts in the
Report Generator. Click the Normalize tab to view the normalization plate map, unit, and scale factor as entered in Wave 2.3
(Figure 6b). Use the Normalize button on the Summary Printout page to toggle the data displayed on each chart between
normalized and non-normalized rate data (Figure 6c).
Note: To preserve data integrity between Wave 2.3 and Report Generators, normalized data exported to a Report Generator is locked for editing. To modify the
normalization values used in the Report Generator, they first must be edited in Wave 2.3 and then re-exported to the Report Generator. If result data has not
been normalized, the Report Generator will not display the Normalize tab.
Figure 6a | Example of a normalization plate
m
ap for the Agilent Seahorse XFp Analyzer in
Wave 2.3.
Figure 6b | The same normalization plate map from Wave 2.3 (Figure
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a) displayed in the Report Generator.
Figure 6c | Normalize button on the Summary
Printout
tab. By default, normalized rate data
exported from Wave
will be displayed
as indicated by
the
Normalize ON button status (green). Click the
Normalize ON
button to toggle the data display to
show
non-normalized rate data (Normalize OFF –
red)
. Data exported from Wave 2.3 without
normalized rate data
will show a gray Normalize
button.
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Exclude Assay Wells (Outliers) or Groups from Report Generator Analysis:
Individual assay wells or entire groups/conditions can be excluded
from parameter calculations in the Report Generator. Before exporting
data from Wave 2.3, click the assay well(s) on the plate map or
double-click the group name(s) on the Group List to exclude those
wells or groups from data export. The Project Information tab displays
the plate map layout and any assay wells or groups that have been
excluded from the Excel export (Figure 7).
Frequently Asked Questions
What rate measurements are used to calculate the parameters in this Report Generator?
Parameter equations are described in Table 1
of this User Guide.
How do I remove outlier wells in the Report Generator?
Outlier assay wells must be turned OFF or reassigned in Wave 2.3 prior to Excel export. See section called
Exclude
Outliers/Groups from Analysis or the Wave User Guide for more information.
Can I use the Excel file exported from the XFp, XF96, or XF24 Analyzer?
It is recommended to export assay result data directly to the XF Report Generator from Wave 2.3 (Desktop or Controller),
however data can also be manually imported as an Excel file (*.xlsx). Excel files exported from earlier versions of Wave (Desktop
or Controller) and XF96/XF24 software are not compatible. If the Excel file has been exported from Wave but cannot be imported
to the Report Generator, please contact Seahorse Technical Support
.
If you receive an error message about Instrument Protocol (XFe96; XFe24; XF96; XF24 only)
Errors upon data import into the Report Generator are likely caused by a custom cycle in the Instrument Protocol. A Custom
Cycle refers to an additional ‘Mix’ or ‘Wait’ command step in the Instrument Protocol an assay. Custom Cycles are not part of
the standardized assay template for the Seahorse XF Cell Mito Stress Test and not supported in Report Generator analysis.
Please contact Seahorse Technical Support
if you have any additional questions regarding Custom Cycles.
Can I use baseline rate data (%) to calculate assay parameters?
Normalized (or non-normalized) OCR data is used for parameter calculations, parameter calculations using baseline rate data
(%) is not supported at this time.
Where is my ECAR data?
ECAR (Extracellular Acidification Rate) data is displayed on the Measures Sheet tab in the Seahorse XF Cell Mito Stress Test
Report Generator.
How do I analyze multiple result files in a Report Generator?
The Seahorse Multi-File XF Cell Mito Stress Test Report Generator enables the import and analysis of multiple XF Cell Mito
Stress Test assay result files. Find out more and download the Multi-File XF Report Generator here
.
Figure 7 | XFp Plate Map on the
Project Information
tab. Assay well C
has been turned off in Wave 2.3 prior
to export, therefore the
Control group
parameter calculations are based on
assay wells B and D only.
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Feedback
Feedback for the Report Generator or other products is always encouraged. Please direct any questions, concerns or
suggestions to Seahorse Technical Support at: seahorse.support@agilent.com
S7888-10001 RevB
RA.4773611111
For Research Use Only. Not for use in diagnostic procedures.
Learn more about Agilent Seahorse XF technology at www.agilent.com
© Agilent Technologies, Inc. 2016, 2020
Published in USA, October 2, 2020
S7888-10001 Rev B
Glossary
Oxygen Consumption Rate (OCR)
The rate of decrease of oxygen concentration in the assay medium. OCR is a measure of the rate of mitochondrial
respiration of the cells.
Extracellular Acidification Rate (ECAR)
The rate of increase in proton concentration [or decrease in pH] in the assay medium. OCR is a measure of the rate of
glycolysis of the cells.
Basal Respiration
Oxygen consumption used to meet cellular ATP demand and resulting from mitochondrial proton leak. Shows energetic
demand of the cell under baseline conditions.
ATP Production
The decrease in oxygen consumption rate upon injection of the ATP synthase inhibitor oligomycin represents the
portion of basal respiration that was being used to drive ATP production. Shows ATP produced by the mitochondria that
contributes to meeting the energetic needs of the cell.
H+ (Proton) Leak
Remaining basal respiration not coupled to ATP production. Proton leak can be a sign of mitochondrial damage or can
be used as a mechanism to regulate the mitochondrial ATP production.
Maximal Respiration
The maximal oxygen consumption rate attained by adding the uncoupler FCCP. FCCP mimics a physiological “energy
demand” by stimulating the respiratory chain to operate at maximum capacity, which causes rapid oxidation of
substrates (sugars, fats, amino acids) to meet this metabolic challenge. Shows the maximum rate of respiration that the
cell can achieve.
Spare Respiratory Capacity
This measurement indicates the capability of the cell to respond to an energetic demand as well as how closely the cell
is to respiring to its theoretical maximum. The cell’s ability to respond to demand can be an indicator of cell fitness or
flexibility.
Non-mitochondrial Oxygen Consumption
Oxygen consumption that persists due to a subset of cellular enzymes that continue to consume oxygen after rotenone
and antimycin A addition. This is important for getting an accurate measure of mitochondrial respiration.
