How is the mitoATP Production Rate calculated if there are different oxidizable substrates (with different P/O ratios) in the assay media?
To convert ATP-coupled respiration (OCR) into mitochondrial ATP production rates, the stoichiometry of ADP phosphorylated to ATP per atom of oxygen reduced at the terminal point of electronic transport chain must be determined. The maximum theoretical P/O value varies between different fuels and depends on the stoichiometry/pathway of the fuel oxidation, as well as the efficiency of the F1F0 ATP synthase. Under standard XF experimental conditions, cells are provided with a mixture of fuels (predominantly glucose, pyruvate and glutamine), and often endogenous fuel stores (glycogen, fatty acids, other amino acids) are available for mitochondrial oxidation. Thus, it was tested with several cell lines and validated that a P/O value of 2.75 accurately represents the average P/O ratio of the mixture of exogenous and endogenous oxidizable substrates. For more information, see the white paper: "Quantifying Cellular ATP Production Rate Using Agilent Seahorse XF Technology" at White Paper Quantifying Cellular ATP Production Rate.
When an Induced XF Real-Time ATP rate assay is performed, how is the Induced rate calculated and reported?
During an XF Real-Time ATP Rate Induced Assay, the Induced Rate(s) displayed in the Summary Printout and Average Assay Parameter Calculations of the Measure Sheet are calculated as the average rate of all the measurements between the acute injection and the oligomycin injection. However, induced rates at each time point can be obtained from the Kinetic Rate Data (Measure Sheet).
What is the XF ATP Rate Index as defined the in XF Real-Time ATP Rate Assay?
The XF ATP Rate Index is the ratio of the mitoATP Production Rate to the glycoATP Production Rate (i.e. mitoATP rate / glycoATP rate). This ratio represents a quantitative metric of the cellular metabolic phenotype. Metabolic indexes greater than 1.0 represent cellular metabolism where greater than 50 % of cellular ATP is derived from mitochondrial through ETC/OXPHOS, while indexes less than 1 indicate greater than 50 % of total ATP is produced by the glycolytic pathway. Since the metabolic index is a ratio-metric measurement, it is highly sensitive to changes or shifts in metabolic phenotype.
Why is the XF ATP Rate different for the same cell type when the cells are plated at different densities?
The metabolic phenotype of the cell will be influenced by the cellular demand for ATP. Cells that are proliferating or differentiating have, in general, higher glycolytic rates than cells that are confluent (slow growing) or terminally differentiated. To compare results between experiments, it is recommended to maintain consistent cell culture conditions and cell seeding densities throughout the investigation.
Why must Seahorse XF DMEM Medium, pH 7.4 or Seahorse XF RPMI Medium, pH 7.4 be used to perform the assay?
The calculation of the glycoATP Production Rate requires an absolute measurement of the glycolytic proton efflux rate during the XF Real-Time ATP Rate Assay. To properly calculate PER, the assay media must have a fixed buffer capacity. A low concentration of HEPES in the media provides consistent buffer capacity values across the time frame of the assay. Although addition of HEPES buffer reduces the ECAR signal slightly, it significantly improves the quality and consistency of the ECAR data, as well as the accuracy of the transformation to PER (For more details see white paper: Improving Quantification of Cellular Glycolytic Rate Using Agilent Seahorse XF Technology. Also, the use of XF DMEM or XF RPMI medium with the pH pre-adjusted to 7.4 saves time in assay preparation and ensures a consistent assay media pH value across XF experiments.
Can I use other Seahorse XF assay media to run the XF Real-Time ATP Rate Assay?
Calculation of accurate glycoATP production rates requires the use of an assay medium without phenol red or bicarbonate and a low concentration of HEPES buffer. Thus, the use of Agilent Seahorse XF DMEM (or RPMI), pH 7.4 is recommended. Any deviation from the recommended medium and supplements (glucose, pyruvate, glutamine) will require that the Buffer Factor should be determined empirically (using the XF Analyzer) for each assay (See the Seahorse XF Buffer Factor Protocol for further information). Any assay medium containing phenol red cannot be used for the XF Real-Time ATP Rate Assay.
When running an induced XF Real-Time ATP Rate Assay, the OCR after oligomycin injection is higher than the basal OCR, what happened?
Adding compounds that uncouple electron transport from oxidative phosphorylation (like FCCP, DNP, etc.) before the oligomycin injection will typically result in an increase in OCR. However, this respiration is not coupled to ATP production since the mitochondrial membrane potential is decreased or dissipated without participation of the ATP synthase, so little to no ATP is generated under these circumstances. In these cases, the basal mitoATP Production Rate cannot be calculated accurately. If an uncoupler compound is suspected, then it is recommended to include a control group with an injection of assay medium + vehicle to calculate basal mitoATP Production Rates.
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Keywords: ATP Rate Assay Kit, ATP Rate, mitoATP Production Rate, P/O ratio, ATP Rate Index, oligomycin, HEPES, Seahorse, XFe96, XFe24, XFp, FAQ