pH-Xtra™ Glycolysis Assay is an easy to use, highly flexible 96 or 384-well fluorescence plate reader-based approach for the direct, real-time, kinetic analysis of extracellular acidification rates (ECA / ECAR). As lactate production is the main contributor to this acidification, ECA measurements are a convenient and informative measure of cellular glycolytic flux. Such measurements offer an important insight into the central role played by altered glycolytic activity in a wide array of physiological and pathophysiological processes, including cellular adaptation to hypoxia and ischemia, and the development and progression of tumorigensis.
The pH-Xtra™ reagent is chemically stable and inert, water soluble and cell impermeable. It exhibits a positive signal response (increased signal with increased acidification) across the biological range (pH6 – 7.5), which coupled with its spectral and response characteristics, make pH-Xtra™ the ideal choice for flexible, high-throughput assessment of ECA. This performance facilitates sensitive robust microtitre-plate based measurements, thereby overcoming many of the problems associated with the more cumbersome potentiometric pH approach. Rates of extracellular acidification are calculated from changes in fluorescence signal over time and as the measurement is non-destructive and fully reversible (pH-Xtra™ reagent is not consumed), measurement of time courses and multiple drug treatments are possible.
Luxcel’s flexible plate reader format also allows multiparametric or multiplex combinations with Luxcel’s other products, and with other commonly available reagents, thereby facilitating parallel kinetic measurements of parameters such as ECA, mitochondria membrane potential (ΔѰm), O2 consumption or ROS generation, followed by the end point measure of parameters such as ATP content or cell membrane integrity, all on the same test cells. For example, the combination of Luxcel’s MitoXpress Xtra® – Oxygen Consumption Assay [HS Method; Cat No. MX-200] and pH-Xtra™ Glycolysis Assay allows the simultaneous real-time measurement of the interplay between mitochondrial respiration and glycolysis. This facilitates the determination of a cell’s metabolic phenotype and the quantification of perturbations in the balance between glycolysis and oxidative phosphorylation under various stimuli or pathological states.
PLATE READER SET-UP
pH-Xtra™ reagent is a chemically stable and inert, cell impermeable H+ sensing fluorophore.
INSTRUMENTS AND SETTINGS
Two fluorescence modalities can be optimally used with the pH-Xtra™ Glycolysis Assay, depending on plate reader type and instrument setup, as follows:
- Standard: Time-resolved fluorescence measurement (TR-F), and
- Advanced: Dual-read Ratiometric TR-F measurement (Lifetime Calculation).
pH-Xtra™ Glycolysis Assay may also be used in non TR-F intensity mode on some plate readers, although we recommend running the Signal Optimisation protocol and optimising cell seeding density.
NOTE: Further details, including instrument, filter selection and measurement settings can be found in Instrument Settings.