JC-1 Fluorescent Mitochondrial Membrane Depolarization Assay

1. Prepare samples and controls, 2. Dilute 10X Apoptosis Wash Buffer 1:10 with diH20., 3. Reconstitute FLICA with 50 μL DMSO., 4. Dilute FLICA 1:5 by adding 200 μL PBS., 5. Add diluted FLICA to each sample at 1:30 (e.g., add 10 μL to 290 μL of cultured cells)., 6. Incubate approximately 1 hour., 7. Remove media and wash cells 3 times: add 1X Apoptosis Wash Buffer and spin cells., 8. If desired, label with additional stains, such as Hoechst, Propidium Iodide, 7-AAD, or an antibody., 9. If desired, fix cells., 10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM FLICA excites at 492 nm and emits at 520 nm., , If working with adherent cells, please see the manual for additional protocols.