Agilent Seahorse Mito Fuel Flex Report Generator FAQs

Document created by courtneynadeau Employee on Oct 15, 2019Last modified by courtneynadeau Employee on Oct 17, 2019
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Why is the scale always 100%?

Absolute OCR (pmol/min) values are transformed into % values for dependency, capacity and flexibility based on their respective definitions and mathematical operations. Thus the theoretical maximal dependency on a single fuel would be 100%.


Should my Dependency values add up to 100%?

If a cell has any flexibility regarding alternative fuels it may oxidize to meet energy requirements, then dependencies for all fuels will not equal 100%.


If I have negative flexibility values, what does it mean?

When changes in OCR are small, well to well variability might lead to negative flexibility values. Negative flexibility values of less than 5% are generally attributable to noise in the assay. If you detect significant negative flexibility, contact Technical Support at: 


What if all three inhibitors only cause a small decrease in total OCR?

Processes other than oxidation of these three fuels may contribute to baseline OCR. These processes may be broken down further into mitochondrial and nonmitochondrial oxygen consuming processes:

  • Other mitochondrial respiration: respiration dependent on alternative substrates being oxidized to support mitochondrial respiration, which may include (but not limited to) short and medium chain fatty acids and amino acids other than glutamine.
  • Nonmitochondrial oxygen consumption: consumption of oxygen by other biochemical processes in the cell. This includes (but is not limited to) very long chain fatty acids that get partially oxidized in the peroxisomes and other cellular enzymatic processes that consume oxygen. The non-mitochondrial fraction of total oxygen consumption can be measured using the Seahorse XF Cell Mito Stress Test.


How do I remove outlier wells in the Report Generator?

Outliers must be removed before data analysis in the Report Generator. See section Excluding Outliers for more information.


I’m unable to import my data as an Excel file into the Report Generator for analysis, why?

Assay result data must be exported from Wave (Desktop or Controller) or from XFp 1.1 software as an Excel Workbook file format (*.xlsx) before importing into any Report Generator. If the Excel file has been exported from Wave but cannot be imported to the Report Generator, please contact Seahorse Technical Support at:


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 your Instrument Protocol. A Custom Cycle refers to an extra ‘Mix’ or ‘Wait’ command step in the Instrument Protocol an assay. Custom Cycles are not part of the standardized assay template for the XF Cell Energy Phenotype Test and are not supported in Report Generator analysis. Please contact Seahorse Technical Support if you have any additional questions regarding Custom Cycles.


How do I combine multiple result files in this Report Generator?

The XF Mito Fuel Flex Test Report Generator enables analysis of an individual assay result file. Combining multiple result files in the Report Generator is not supported currently.


What rates are used to calculate Dependency, Flexibility and Capacity?

Rates used for calculations are described on page 4. The Baseline Rate is defined as the last rate measurement before the FIRST injection. Dependency and Capacity Rates are defined as the LAST rate measurement before the NEXT injection.  See Parameter Calculations for more information about calculations in the XF Mito Fuel Flex Test Report Generator.


Will these inhibitors and concentrations work with all cells?

Yes, the test uses all three compounds at concentrations well above their EC50 values for inhibition in mammalian cells. These values have been validated in many cell lines and primary isolates. While most cell types or cell lines have an appreciable response to at least one inhibitor, not all cells will respond to all inhibitors. If the cells are not responsive to a particular inhibitor, they may not depend on that particular fuel pathway (i.e., they are flexible with respect to the fuel used for oxidative phosphorylation).


How do I interpret ECAR and glycolysis in this assay?

Using combinations of inhibitors can confound interpretation of ECAR data with this test due to shifts in cellular ATP production and demand. For directly measuring glycolytic function, we recommend using the Seahorse XF Glycolysis Stress Test.


The recommended assay medium doesn’t include fatty acid; can I add it?

Although not required, long chain fatty acid may be added to the medium. We recommend using a single species of long chain fatty acid, such as Seahorse XF palmitate-BSA FAO substrate, when testing exogenous fatty acid oxidation. NOTE: only oxidation of long-chain fatty acid, such as palmitate, is sensitive to inhibition by etomoxir.


More FAQs: Cell Analysis FAQs and Troubleshooting


Keywords: Mito Fuel Flex, Dependency, Capacity, Flexibility, Fatty Acid, Negative Flexibility, Report Generator, Report Generator Error, Parameter Equations, Outlier, Seahorse, XFe96, XFe24, XFp, XF24, XF96, FAQ