Signal Optimisation – recommended for first time users
NOTE: Use a plate block heater fro plate preparation and pre-warm plate reader to measurement temperature.
STEP 1: Reconstitute contents of the MitoXpress® Xtra vial in 1ml of water, PBS or culture media, gently aspirating 3-4 times. 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).
STEP 2: Prepare 8 replicate wells of a 96-well plate, by adding 150µl pre-warmed culture medium to each well (A1-A4, B1-B4).
STEP 3: Add 10µl reconstituted MitoXpress® Xtra reagent to 4 of the replicate wells (A1-A4) and 10µl water, PBS or media to the remaining replicate wells (B1-B4).
STEP 4: Promptly add two drops (or 100µl) pre-warmed HS Mineral Oil to all eight replicate wells, taking care to avoid air bubbles. NOTE: See Appendix B in User Manual – HS Mineral Oil Pipetting Tips.
STEP 5: Read plate immediately in a fluorescence plate reader over 30 minutes (read every 2-3 minutes).
STEP 6: 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 plate readers, set up according to Appendix A in the User Manual – Instrument Settings, MitoXpress® Xtra should return a S:B≥3. Higher readings are expected with TR-F and dual read TR-F measurement. NOTE: See also Appendix C in User Manual – Trouble Shooting.