NOTE: We recommend that all first time user perform a Signal Optimisation test. Signal and Blank Control wells may also be included.
Assessing Oxygen Consumption
Plot the Blank Control well-corrected MitoXpress® Xtra Intensity or Lifetime values versus Time (mins; Figure 7). Select the linear portion of the signal profile (avoiding any initial lag or subsequent plateau) and apply linear regression to determine the slope (OCR) and correlation coefficient for each well. NOTE: This approach is preferable to calculating a slope from averaged profiles.
Tabulate the slope values for each test sample, calculating appropriate average and standard deviation values across replicate wells. If optional Signal Control wells are included, the slope obtained for the Signal Control (sample without cells) should be subtracted from all test values. Data analysis templates are available from some plate reader manufacturers, specifically configured to automate the analysis of Luxcel’s MitoXpress® range of assays.
Figure 7: Typical Lifetime profile of MitoXpress® Xtra for adherent cells, treated with different ETC compounds, including Antimycin A (recommended as a Negative Control). The effect of Glucose Oxidase as a positive Signal Control is illustrated schematically. NOTE: If using FCCP it is strongly recommended to perform a dose titration, since FCCP exhibits a bell shaped response.
Plotting a Dose Response Curve
To generate a dose response curve, plot the data generated as outlined above against the corresponding compound concentration (Figure 8).
Figure 8: The dose response curve presented here is an example of the data typically produced with this assay. Drug concentration (µM) versus calculated slope (µs / hour) demonstrates that this drug causes inhibitory response on cellular respiration.
Cellular Energy Flux Analysis
Multiparametric (or multiplex) combination of MitoXpress® Xtra – Oxygen Consumption Assay [HS Method] together with Luxcel’s pH-Xtra® – Glycolysis Assay (Cat No: PH-200) allows the simultaneous real-time measurement of mitochondrial respiration and glycolysis and analysis of the metabolic phenotype of cells and the shift (flux) between the two pathways under pathological states (Figure 9)
Figure 9: Cellular Energy Flux for HepG2 cells, treated with a combination of drug compounds modulating the ETC or inhibiting lactate production, shown as a percentage relative to untreated control cells. Comparative measurements with MitoXpress® Xtra and pH-Xtra™, show the shift between mitochondrial respiration and glycolysis and the cellular control of energy (ATP; measured 1h post-treatment using Promega Cell Titer-Glo®).