SIGNAL OPTIMISATION – recommended for first time users
NOTE: Use a plate block heater for plate preparation and pre-warm plate reader to measurement temperature.
STEP 1: Reconstitute Respiration Buffer table in 50ml of water, warm to assay temperature (37°C), pH adjust to approx. pH7.4 and filter sterilise using a 0.22µm filter. Reconstitute (transparent) contents of the pH-Xtra™ vial in 1ml of 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 aliquotes in water -20°C for use within one month (avoid freeze thaw).
STEP 2: Prepare 8 replicate wells of a 96-well plate, by adding 150µl pre-warmed Respiration Buffer to each well (A1-A4, B1-B4).
STEP 3: Add 10µl reconstituted pH-Xtra™ reagent to 4 of the replicate wells (A1-A4) and 10µl Respiration Buffer to the remaining replicate wells (B1-B4).
STEP 4: Read plate immediately in a fluorescence plate reader over 30 minutes (read every 2-3 minutes).
STEP 5: Examine Signal Control well (A1-A4) and Blank Control well (B1-B4) readings (linear phase) and calculate S:B ratio. NOTE: For dual-read TR-F, calculate S:B for each measurement window.
For most fluorescence TR-F plate readers, set up according to Instrument Settings, pH-Xtra™ should return a S:B≥3.