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

Protocol summary

1. Prepare cells
2. Add test compounds
3. Add MitoLite™ NIR working solution (100 µL/well/ 96-well plate or 25 µL/well/384-well plate)
4. Incubate at 37°C, 5% CO₂ incubator for 30 - 60 minutes
5. Add Assay Buffer B (50 µL/well/96-well plate or 12.5 µL/well/384-well plate)
6. Monitor fluorescence intensity (Bottom read mode) at Ex/Em = 640/680 nm (Cutoff = 665 nm)

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

Add 50 µL of 200X MitoLite™ NIR (Component A) into 10 mL of Assay Buffer A (Component B) and mix well to make MitoLite™ NIR 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.
   
   For Negative Control: Treat cells with vehicle only.
   
   For Positive Control: Treat cells with FCCP or CCCP at 5 - 50 µM in a 37°C, 5% CO₂ incubator for 15 to 30 minutes. Note: CCCP or FCCP can be added simultaneously with MitoLite™ NIR. To get the best result, titration of the CCCP or FCCP may be required for each individual cell line.
   
   
2. Remove the cell medium before adding MitoLite™ NIR working solution. Note: It is important to remove the cell medium before adding MitoLite™ NIR working solution.
   
   
3. Add 100 µL/well/96-well plate or 25 µL/well/384-well plate of MitoLite™ NIR working solution into the cell plate.
   
   
4. Incubate the plate in a 37°C, 5% CO₂ incubator for 30 - 60 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.
   
   
5. 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 before monitoring the fluorescence signal. 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).
   
   
6. Monitor the fluorescence intensity with a fluorescence microplate reader (bottom read mode) at Ex/Em = 640/680 nm (Cutoff = 665 nm) either using the endpoint mode or using the kinetic mode 10 to 30 minutes after adding Assay Buffer B (Component C).