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 an alternative substrate(s) being oxidized to support mitochondrial respiration. This 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 nonmitochondrial fraction of total oxygen consumption can be measured using the Seahorse XF Cell Mito Stress Test.
Why is Dependency reported as zero?
If Dependency is not significantly above zero or negative due to well to well variability, there is no dependency on that particular substrate. If the cells are not dependent on the target fuel pathway, OCR may slightly increase following injection of inhibitor. When this occurs, Dependency is automatically set to zero (no dependence), and Flexibility will be equal to Capacity.
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.
How can I further diagnose or troubleshoot Mito Fuel Flex Test results?
The preceding potential issues described (apparent low response to inhibitors, 0 % dependency, or negative flexibility values) may be further diagnosed by performing (as a separate test or added group) a Mito Fuel Flex Test with only media injections (no inhibitors) to establish if baseline respiration changes significantly over the course of the assay. If the absolute respiration rates (OCR) in the absence of inhibitors trend significantly upward or downward throughout the assay, then the parameters of this test (relative % dependency, capacity and flexibility) will be either underestimated or exaggerated, respectively. This depends on the magnitude and direction of any baseline trends versus the magnitude of change due to added inhibitors. To limit any upward/downward baseline trending OCR, ensure that cell culture and technical parameters of the assay have been thoroughly optimized.
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 (that is, they are flexible regarding 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 does not 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
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