Cell Meter™ Mitochondrion Membrane Potential Assay Kit *Red Fluorescence Optimized for Microplate Reader*

Protocol summary

1. Prepare cells
2. Add test compounds
3. Add MitoTell™ Red working solution (100 µL/well/ 96-well plate or 25 µL/well/384-well plate)
4. Incubate the plate in a 5% CO₂, 37°C incubator for 30 minutes
5. Add Assay Buffer B (50 µL/well/96-well plate or 12.5 µL/well/384-well plate) 
6. Monitor the fluorescence increase (bottom read mode) at Ex/Em = 610/650 nm (Cutoff = 630 nm) or fluorescence microscope with Cy5 filter

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

Add 50 µL of 200X MitoTell™ Red (Component A) into 10 mL of Assay Buffer A (Component B) and mix well to make MitoTell™ Red working solution. Protect from light.

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

1. Treat cells with test compounds for a desired period of time to induce apoptosis, and set up parallel control experiments. Note: We treated HeLa cells with 20 µM CCCP for 15 minutes to change the mitochondrial membrane potential. See Figure 1 for details. CCCP or FCCP can be added simultaneously with MitoTell™ Red. To get the best result, titration of the CCCP or FCCP may be required for each individual cell line.
   
   
2. Add 100 µL/well/96-well plate or 25 µL/well/384-well plate of MitoTell™ Red working solution into the cell plate.
   
   
3. Incubate the plate at 37ºC for 15 - 30 minutes, protected from light. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
   
   
4. Add 50 µL/well/96-well plate or 12.5 µL/well/384-well plate of Assay Buffer B (Component C) into the cell plate. Note: DO NOT wash the cells after loading. For non-adherent cells, it is recommended to centrifuge cell plates at 800 rpm for 2 minutes with brake off after adding Assay Buffer B (Component C).
   
   
5. Monitor the fluorescence intensity with a fluorescence microplate reader (bottom read mode) at Ex/Em = 610/650 nm (Cutoff = 630 nm) 10 to 30 minutes after adding Assay Buffer B (Component C) or observe the fluorescence signal under a fluorescence microscope with Cy5 filter set.