CELL CULTURE AND PLATING
NOTE: Always leave two well (H11 and H12) free from the addition of MitoXpress® Xtra reagent, as Blank Controls.
- For Adherent cells, seed cells in a 96-well plate at a density (typically 40,000 – 80,000 cells / well) in 200µl culture medium. Incubate overnight in a CO2 incubator at 37°C.
- For Suspension cells, seed on the day of assay in 150µl culture medium at a density of ∼4 x 106 / ml.
- Reconstitute the contents of the MitoXpress® Xtra vial in 1ml of water, PBS or culture media, gently aspirating 3-4 times (Figure 3). NOTE: Reconstituted probe stock 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 Antimycin A (Complex III inhibitor), FCCP (ETC uncoupler) and Glucose Oxidase (GOx: positive 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 Oxygen Consumption or to investigate the effect of a compound on electron transport chain function (ETC; oxidative phosphorylation), 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 replace with 150µl of fresh culture media (Figure 4). NOTE: We recommend always leaving two wells (H11 and H12) free from the addition of MitoXpress® Xtra reagent, for use as Blank Controls. Add 150µl of fresh culture media to these Blank Control wells also.
STEP 2: Add 10l reconstituted MitoXpress® reagent to each well, except those wells for use as Blank Controls. Add 10µl of fresh culture media to these Blank Control wells. NOTE: If plating a full 96 well plate of assays, we recommend combining Step 1 and Step 2 by adding the 1ml of reconstituted MitoXpress® Xtra reagent to 15ml pre-warmed fresh culture media and using a multi-channel pipette to add 150µl of MitoXpress® Xtra in media stock to each well (Figure 4). Add 150µl of fresh culture media only (no MitoXpress® Xtra) to the Blank Control wells.
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: Promptly seal each well by adding two drops (or 100µl) pre-warmed HS Mineral Oil, taking care to avoid air bubbles (Figure 5). NOTE: Small variations in the volume of oil (between 90-110µl) should not adversely affect the readings using MitoXpress® Xtra.
STEP5: Read the plate immediately in a fluorescence plate reader, with the set-up as described in Instrument Settings (Figure 6). The plate should be measured kinetically for >90 minutes. When 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 Controls. Add 150µl of fresh culture media +10µl of reconstituted MitoXpress® Xtra reagent to each well.
Positive Controls: Leave 2 or 3 wells free from the addition of cells for use as Positive Controls. Add 150µl of fresh culture media +10µl of (1mg/ml) Glucose Oxidase stock solution (in water) + 10µl reconstituted MitoXpress® Xtra reagent to each well.
Negative Controls: To 2 or 3 wells containing cells, add 1µl of (150 µM) Antimycin A stock solution (in DMSO) + 10µl reconstituted MitoXpress® Xtra reagent.