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XF Stress Test Report Generator

User Guide

Copyright 2014

103247-400 Rev. B
XF Stress Test Report Generator User Guide

Table of Contents

Introduction ................................................................................................................................................................................3

I. Configure Excel to Enable Macros ..................................................................................................................................... 3

II. Save Wave Output file in Excel format (*.xlsx) ........................................................................................................................ 3

III. Load Excel File into XF Stress Test Report Generator ............................................................................................................. 4

IV. Customize and Review XF Stress Test Report ................................................................................................................... 5

Advanced Options.......................................................................................................................................................................5

Outlier Wells ...............................................................................................................................................................................6

Assay Parameter Calculations ............................................................................................................................................................ 7

XF Stress Test Report Generator User Guide

Introduction
XF Stress Test Report Generators enables XF users to generate a customizable, convenient summary of either XF Stress
Test assay. The values provided by the XF Stress Report Generators are calculated as absolute oxygen consumption rate
(OCR) in pmol O2/min or absolute extracellular acidification rates (ECAR) in mpH/min (and therefore, does not use
Area-Under-the-Curve (AUC) calculations).
Workflow Diagram

I. Configure Excel to Enable Macros

Both the XF Cell Mito Stress Test Report Generator (102990-200_RA.xlsm) and XF Glycolysis Stress Test Report
Generator (102991-200_RA.xlsm) require Microsoft Excel 2007, 2010, or 2013. Note: Excel must be configured
to allow macros to run. Select Enable Editing (Located on the yellow information bar) if prompted to do so when
opening the Report Generator file for the first time.
To enable macros: Launch Excel File Options Trust Center Trust Center Settings
Macro Settings Select Enable all macros or Disable all macros with notification or Disable all macros
except digitally signed macros
Message Bar Settings Select Show the message bar in all applications when active content, such as
ActiveX controls and macros, has been blocked
Note: XF Stress Report Generators require the Measure after Injection box to remain checked in the assay design
file (which is the default setting) to yield correct parameter calculations.

II. Save Wave Output file in Excel format (*.xlsx)

1.

Open the XF Stress Test Assay results file (*.asyr) in Wave.

Note: *.xfd files will need to be converted to *.asyr before using the XF Stress Test Report Generators.
To convert these files: Open the Wave program, browse and select the .xfd result file, then click Open.
Once the result file is open, click Save as and select Assay Analyze (*.asyr) as the file type, rename
(if necessary) and select location to save the file.
Once the .xfd file has been saved as an *.asyr file, proceed to step 2.

2.

Once the result file (*.asyr) is open, click Save As. Select Excel 2007/2010 (*.xlsx) as the file type and
follow the prompts to name and select a location to save the Excel output file.

XF Stress Test Report Generator User Guide

Double-click on the desktop icon to open either the XF Cell Mito

Stress Test or XF Glycolysis Stress Test Report Generator (as
dictated by the XF Stress Test being analyzed).

2.

Click Launch Tool to load the newly created Excel output file
and run the Report Generator.

OCR (pMoles/min)

OCR (pMoles/min)

1.

OCR (pMoles/min)

III. Load Excel File into XF Stress Test Report Generator

Basa l

Spa re Res pira tory Ca pa city

P roto n Le a k

A T P P roduction

N ot e s :

3.

Click Yes to select a new data file for analysis.

4.

Browse and select the newly created Excel file (from Section II)

5.

In the Load Options window, select which groups to display in the

report. The order in which the groups are displayed can be
altered from the default by selecting the group and clicking
either the Move Up or Move Down button.

6.

Click the Advanced button to view measurement summary and Error Bar type. The application automatically
populates the Advanced Options fields based on the assay run. Refer to Section IV- Advanced Options for more
details.

7.

Click Update Summary

then click OK when the Success! Window appears.

Optional Normalization Steps:

8.

Select normalization data to apply to Report Generator data.

a. Wave Desktop - Open an Assay Result file (.asyr) and from the Overview page, click on Modify > Normalization.
b. Microsoft Excel Open Excel file > Select data to input > Right-click and select Copy.

9.

Click Select All.

10. In the Report Generator, select the Normalization tab at the bottom of the program.
11. Right-click and select Paste.
12. Click Normalize Data.
13. In the Load Options window, select which groups to display in the report. The order in which the groups
are displayed can be altered from the default by selecting the group and clicking either the Move Up or
Move Down button.
14. Click Update Summary.
15. Click OK.

XF Stress Test Report Generator User Guide

IV. Customize and Review XF Stress Test Report

1.

Each report includes three tabs: Summary Printout, Bar Charts, and Measures.
a) Summary Printout is a one-page graphical summary of the XF assay data.
b) Bar Charts provide a graphical representation of all the calculated parameters.
c) Measures provides all the calculated parameters of the stress test (including errors), a kinetic graph of either
ECAR for the XF Cell Mito Stress Test or OCR for the XF Glycolysis Stress Test, and the raw data used to
generate the information in the Bar Charts and the Summary Printout tabs.

2. Customize the charts if desired, then click Save As. Rename and select a location to save the file.
Refer to Section V for Assay Parameter Calculation discussion.

Advanced Options
The XF Stress Test Report Generator uses the data in the Excel Wave Output file to automatically
populate the Advanced Options fields. If any measurements were incorrectly assigned in the
XF Stress Report Generators, corrections can be made in Advanced Options.
a)

Acute injection
An injection that occurs in the assay protocol following the basal measurement, but
before oligomycin in the XF Cell Mito Stress Test or glucose injection in the XF Glycolysis
Stress Test, respectively, and must be injected via Port A.
Note: Acute injections of test compounds in B, C, or D will result in inaccurate XF stress
test parameter calculations.
If the box is checked (by default when four injections are included in the assay protocol), the Report Generator
assumes that an acute injection was part of the assay protocol and the measurements preceding the second
injection will be used to calculate the acute response parameters.

If the box is unchecked (when there are only three injections in the assay protocol), the Report Generator
assumes that there was no acute injection in the assay protocol and the measurements before the first
injection will be used in the basal parameter calculations.
b) Number of Basal and Injection Measurements:
Pre-populated measurements based on instrument protocol.
The number of measurements re-assigned to basal and injection measurements by the user, if required.
c)

Error Bar Info:

Standard Deviation is selected by default (Users may also choose Standard Error of the Mean).
Standard Deviation is calculated using the Microsoft Excel function.
Standard Error of the Mean is calculated using the equation:

( )
( )

Error Bars are calculated from each replicate of the rate measurement used to determine the stress test parameter
(See Tables 1 & 2).

XF Stress Test Report Generator User Guide

Outlier Wells
Outliers or unwanted wells may be excluded from the XF Stress Test Report Generator parameter calculations by reassigning
them from their original group in Wave before saving the file to Excel.
1. From the Wave Overview Page, click Modify in the upper right corner, then select Groups/Conditions.
2. In the Groups/Conditions view, click
to add a new group and name it Outliers. Switch to the Plate Map
view, and select the wells to be included in the Outliers group.
o
o

Click Apply. Select Save As and set the file type to Excel 2007/2010 (*.xlsx).
Load the Wave Excel output file into the XF Stress Test Report Generator.

1.

2.

3.

3. The Wave Excel output file will list the newly created
Outlier group in the Load Options window.
Note: Make sure the Outlier group is unchecked
in the Load Options window so the outliers are
omitted from the calculations.

XF Stress Test Report Generator User Guide

Assay Parameter Calculations

This section describes the how the assay parameters are calculated by the XF Stress Test Report Generators. Values are
calculated as absolute oxygen consumption rate (OCR) in pmol O2/min or absolute extracellular acidification rates (ECAR)
in mpH/min, and use the AVERAGE value of the selected wells within a group to calculate a specific parameter (i.e. the
XF Stress Test Report Generators do not use Area-Under-the Curve (AUC) calculations). Please contact Seahorse Technical
Support with any questions.
1.

XF Cell Mito Stress Test Parameters

Table 1: XF Cell Mito Stress Test Parameters

Parameter

Rate Measurement Equation Used by Report Generator

Non-Mitochondrial Respiration

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 measurement rate before Oligomycin injection) (Last rate measurement before acute
injection)

Coupling Efficiency

(ATP Production Rate) / (Basal Respiration Rate) 100

XF Stress Test Report Generator User Guide

2.

XF Glycolysis Stress Test

Table 2: XF Glycolysis Stress Test Parameters

Parameter

3.

Rate Measurement Equation Used by Report Generator

Glycolysis

(Maximum rate measurement before Oligomycin injection) (Last rate

measurement before Glucose injection)

Glycolytic Capacity

(Maximum rate measurement after Oligomycin injection) (Last rate

measurement before Glucose injection)

Glycolytic Reserve

(Glycolytic Capacity) (Glycolysis)

Glycolytic Reserve as a %

(Glycolytic Capacity Rate) /( Glycolysis) 100

Non-Glycolytic Acidification

Last rate measurement prior to glucose injection

Acute Response

(Last measurement rate before glucose injection Last rate measurement

before acute injection)

Modified XF Stress Test

103247-400 Rev. A

For XF Stress Tests that have been modified to include an acute injection (refer to Section III: Advanced OptionsAcute injection) the points used for parameter calculations are shifted by the number of acute response
measurements taken. For example, if there are 3 acute response measurements, the points used for calculations
are shifted by 3. Should you have any question, please contact Seahorse Technical Support.

XF Stress Test Report Generator User Guide