Mitochondrial Membrane Potential Assay Kit (II) #13296

#13296 Mitochondrial Membrane Potential Assay Kit (II) Protocol

A. Solutions and Reagents

1. 1X PBS: Prepare by diluting 20X PBS (included in each kit, #9808) in reverse osmosis deionized (RODI) or equivalently purified water.

   NOTE: For flow cytometry, adding 0.5% BSA to wash buffer may help to prevent cell loss during the process.

2. TMRE: Add 55 µl DMSO (#12611) to each vial of TMRE to make a 1000µM stock solution. Each vial includes enough TMRE for five 96-well plates (0.1 µl/well) or 50 flow cytometry assays (10 µl/assay). Aliquot if desired and store at -20°C.
   Dilute TMRE to 1:500 with full cell culture medium to make 10X TMRE Labeling Solution. 2 µM is used in this protocol; 0.1 to 10 µM is recommended based on different cell lines.
3. CCCP: If CCCP is used as positive control, allow CCCP solution to equilibrate to room temperature before use.

B. Test Procedure

For Suspension Cells

1. Suspend cells in warm medium or PBS at 1 x 10⁶ cell/ml. Prepare 1 ml aliquots; each 1 ml cell aliquot is one assay point. Make sure there are enough cells for your experiment. For example, if one compound is going to be assayed at three different concentrations, a total of 4 x 1 ml samples will be needed (this includes a positive control).
2. Add test compound(s) to sample tubes at desired concentration and incubate cells for desired time. For best results, a compound titration and incubation time course can help to determine the best assay conditions. To prepare the positive control (mitochondrial membrane potential loss), add 1 µl of 50 mM CCCP (supplied with this kit) to the control tube for a 50 µM final concentration; incubate cells at 37°C for 15 min.
3. Add 100 µl of the 2 µM TMRE Labeling Solution to each sample (200 nM final concentration) and incubate cells in the incubator (37°C and 5% CO₂) for 15 to 30 min.

   Note: 200 nM TMRE is recommended in this protocol. For best results, a titration of TMRE is recommended.

4. Centrifuge sample at 300 g for 5 min, then remove the supernatant.
5. Wash cells once with 1 ml warm 1X PBS wash buffer, repeat step 4.
6. Resuspend cells into 1000 µl warm 1X PBS.
7. Analyze sample on a flow cytometer. If samples are analyzed on plate reader, transfer 100 µl/ cell suspension/well to a black 96-well plate with a clear bottom and read plate on the plate reader. The settings are excitation about 550 nm and emission about 580 nm.

For Adherent Cells

1. Plate cells to a 96-well plate in warm culture medium and culture cells in incubator overnight to allow cells to attach to the plate. A typical cell number is between 1-5 x 10⁴ cells/ well. A cell number titration may be necessary for optimal results.
2. Aspirate the medium from the plate and add test compounds in growth medium or 1X PBS to plate at 100 µl/well at desired concentration and incubate cells for desired time. Compound titration and incubation time course can help to determine the best assay conditions. For positive control (mitochondrial membrane potential loss), add CCCP (supplied with this kit) to the control wells for a 50 µM final concentration and incubate cells at 37°C for 15 min.

   Example: Add 1 µl of 50 mM stock CCCP to 100 µl medium to make 500 µM CCCP; then add 10 µl of this 500 µM CCCP to each well containing 100 µl medium to get final concentration of 50 µM.

3. Add 10 µl of 2 µM TMRE Labeling Solution to each well to get a final concentration of 200 nM and place plate in an incubator (37°C and 5% CO₂) for 20 min.

   Note: 200 nM TMRE is recommended in this protocol. For best results, a titration of TMRE is recommended.

4. Aspirate the solution from the plate.
5. Wash plate 3 times with warm 1X PBS and then add 100 µl/well 1X PBS to the plate.
6. Analyze samples on the plate reader. The settings are excitation about 550 nm and emission about 580 nm.