Screen Quest™ Membrane Potential Assay Kit *Red Fluorescence*

Protocol summary

1. Prepare cells in growth medium or HHBS
2. Add MP dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
3. Incubate at room temperature or 37 ^oC for 1 hour
4. Monitor the fluorescence intensity at Ex/Em = 620/650 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

MP dye-loading solution:
Add 1 mL of 10X MP Sensor (Component A) into 9 mL of HHBS (Component B), and mix well. Note: The MP dye-loading solution is stable for at least 2 hours at room temperature. Note: 1 mL of 10X MP Sensor is enough for one plate. Unused 10X MP sensor (Component A) can be aliquoted and stored at < -20 ^oC for a few months, if stored properly. Avoid repeated freeze-thaw cycles. Note: HHBS (Component B) can be stored at 4 ^oC for convenience.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MP dye-loading solution into the cell plate. Note: If your screening compounds interfere with growth medium and serum factors, replace the growth medium with equal volume of HHBS buffer before adding the MP dye-loading solution. Alternatively, cells can be grown under serum-free conditions. Note: DO NOT wash the cells after dye loading.
   
   
2. Incubate the dye-loading plate in a 5% CO₂, 37 ^oC incubator for 30 to 60 minutes. Note: In some cases, 30 to 60 minutes room temperature incubation may work better.
   
   
3. Prepare the compound plates by using HHBS (Component B) or your desired buffer. Prepare the compound plates with HHBS or the desired buffer.
   
   
4. Monitor the fluorescence intensity at Ex/Em = 620/650 nm (bottom read). Note: It is important to run the signal test before the experiment. Different instruments have their own intensity range. Adjust the signal test intensity to the level of 10% to 15% of the maximum instrument intensity counts.