CELL CULTURE AND PLATING
NOTE: Always leave two wells (H11 and H12) free from the addition of pH-Xtra reagent, as Blank Controls.
- For Adherent cells, seed cells in a 96-well plate at a density (typically 30,000 – 80,000 cells/well) in 200µl culture medium. NOTE: For new cell types, we recommend setting up a titration to select the optimum cell seeding density (see Figure 7).
- For Suspension cells, see on the day of assay in 150µl culture medium at a density of approx. 250,000 – 500,000 cells/well.
PRE ASSAY PREPARATION
NOTE: Where cells are cultured in a CO2 incubator overnight, it is important to purge the media and plasticware of CO2 prior to conducting the pH-Xtra™ Glycolysis Assay as residual CO2 may contribute to acidification. Perform a CO2 purge, by incubating cells in a CO2 free incubator at 37°C with 95% humidity, approx. 3 hours prior to performing the Glycolysis Assay measurement.
- Reconstitute Respiration Buffer tablet in 50ml of water, pH adjust to approx. pH7.4 and filter sterilise using a 0.22µm filter. Reconstitute transparent contents of the pH-Xtra™ vial in 1ml Respiration Buffer, gently aspirating 3-4 times. NOTE: Reconstituted pH-Xtra™ reagent can be stored in the dark between +2 to +8°C for several days or stored as aliquots in water at -20°C for use within one month (avoid freeze thaw).
- Prepare test compounds, controls and dilutions as desired. Typical controls are oxamic acid (inhibitor; decrease ECA), FCCP (ETC uncoupler; increases ECA) and glucose oxidase (GOx; signal control).
NOTE: We recommend that all culture media and stock solutions to be used in the assay are pre-warmed at 37°C prior to use. Use a plate block heater for plate preparation and pre-warm the fluorescence plate reader to measurement temperature.
To assess Extracellular Acidification (ECA) or to investigate the effect of a treatment on glycolytic flux, cells are treated immediately prior to measurement. NOTE: We recommend the use of triplicate wells for each treatment.
STEP 1: Remove spent culture medium from all assay wells and wash cells twice (2X), using 100µl of Respiration Buffer per well for each wash (Figure 4). After removing the second wash, replace with 150µl of fresh Respiration Buffer. NOTE: We recommend always leaving two wells (H11 and H12) free from the addition of pH-Xtra™ reagent, for use as Blank Controls. Add 150µl of Respiration Buffer to these Blank Control wells also.
STEP 2: Add 10µl reconstituted pH-Xtra™ reagent to each well, except those wells for use as Blank Controls. Add 10µl of Respiration Buffer to these Blank Control wells.
NOTE: If plating a full 96-well plate of assays, we recommend simplifying Step 1 and 2 by preparing a stock solution containing the 1ml of reconstituted pH-Xtra™ reagent added to 15ml pre-warmed Respiration Buffer, and using a multi-channel pipette to add 150µl of this diluted pH-Xtra™ stock to each well. Add 150µl of Respiration Buffer only (no pH-Xtra™) to each Blank Control well.
STEP 3: Test compound stock or vehicle (typically 1-10µl) may be added at this point if desired. NOTE: We recommend keeping the volume of added compound low to minimise any potential effects of solvent vehicle.
STEP 4: Read the plate immediately in a fluorescence plate reader, with the set-up as described in the Instrument Settings (Figure 5). The plate should be measured kinetically for >120 minutes. When the measurement is completed, remove the plate and save measured data to file.
- Signal Controls: Leave 2 or 3 wells free from the addition of cells for use as Signal Control wells. Add 150µl of Respiration Buffer + 10µl of reconstituted pH-Xtra™ reagent to each well.
- Positive Signal Controls: Leave 2 or 3 wells free from the addition of cells for use as Positive Control wells. Add 150µl of fresh Respiration Buffer + 10µl of [1 mg/ml] glucose oxidase stock solution [in water] + 10µl reconstituted pH-Xtra™ reagent to each well.
- Negative Controls: To 2 or 3 wells containing cells, washed and refreshed with 150µl of Respiration Buffer, add 10µl of [750 mM] oxamic acid stock solution [in water] + 10µl reconstituted pH-Xtra™.