NOTE: We recommend that all first time users perform a Signal Optimisation test. Signal and Blank Control wells may also be included.
Figure 6: Typical Lifetime profile of pH-Xtra™ for adherent cells, treated with typical control compounds, including oxamic acid 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 recopmmended to perform a dose titration, since GCCP exhibits a bell-shaped dose response.
ASSESSING EXTRACELLULAR ACIDIFICATION
Plot the Blank Control well corrected pH-Xtra™ Intensity or Lifetime values versus Time (Figure 6). Select the linear portion of the signal profile (avoiding any initial lag or subsequent plateau) and apply linear regression to determine the slope (ECA) 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 etmplates are available from some plate reader manufacturers, specifically configured to automate the analysis of Luxcel’s pH-Xtra™ Glycolysis Assay.
TITRATION OF CELL SEEDING DENSITY
To determine an optimal cell seeding density for performing the pH-Xtra™ Glycolysis Assay, for new cell types, seed replicate wells with a range of seeding densities (typically 0, 10,000, 20,000, 40,000, 60,000 and 80,000 cells/well). Plot the data generated as a function of intensity or Lifetime values versus time, as illustrated (Figure 7).
Figure 7: Extracellular Acidification rate profiles (ECA) are shown for A549 cells seeded at 0, 10,000, 20,000, 40,000, 60,000 and 80,000 cells / well. In this experimental example, a seeding density of 40,000 cells / well was chosen for study as this provided a suitable balance between ECA response and cell availability.
CELLULAR ENERGY FLUX ANALYSIS
Multiparametric (or multiplex) combination of pH-Xtra™ Glycolysis Assay together with Luxcel’s MitoXpress® Xtra – Oxygen Consumption Assay [HS Method] (Cat No: MX-200) allows the simultaneous real-time measurement of glycolysis and mitochondrial respiration, and analysis of the metabolic phenotype of cells and the shift (flux) between the two pathways under pathological states (Figure 8).
Figure 8: 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 pH-Xtra™ (glycolysis) and MitoXpress® Xtra (oxygen consumption), show the shif between glycolysis and mitochondrial respiration and the cellular control of energy (ATP; measured 1h post-treatment using Promega Cell Titer-Glo®).
CALIBRATION OF pH-Xtra™ Glycolysis Assay TO A pH [H+] SCALE
It is possible to express Extracellular Acidification (ECA) as a function of pH [H+] versus time. This is achieved by first creating a calibration standard curve, by measuring TR-F intensity or preferably Lifetime values (selecting stabilised readings over a 30 minute read), from a range of pH-buffered standards at the appropriate assay temperature (see Figure 9). Select the linear portion of the standard curve and apply linear regression to determine the calibration function (See Hynes et al., 2009).
Figure 9: pH-Xtra™ reagent calibration in Lifetime scale, at 30°C and 37°C using pH-buffered PBS, at increments of 0.2 across a pH range 6.0 – 7.5.