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.

Mitochondria play a central role in the biochemical processes associated with the life and death stages of eukaryotic cells. Under normal physiological conditions, a membrane-based proton pump generates an electrochemical gradient, enabling the production of ATP to drive cellular energy-dependent processes. The oxidation of glucose and fatty acids by enzymes associated with the mitochondrial respiratory chain establishes a proton and pH gradient across the mitochondrial inner membrane, resulting in a transmembrane electrical potential gradient (∆Ψm) of -80 to -120 mV and a pH gradient of 0.5-1.0 pH units. Depolarization of the inner mitochondrial membrane can lead to an opening of the mitochondrial permeability transition pore (PTP). This results in the leakage of intermembrane proteins, including cytochrome C, that facilitate the induction of apoptosis through apoptosome formation. Caspase activation has been shown to accelerate the process of ∆Ψm loss. Moreover, a feedback mechanism that results in the generation of reactive oxygen species further accelerates the rate of cell death. Because mitochondrial dysfunction has been closely tied to such neurodegenerative diseases as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, mitochondria remain an important organelle of study. Loss of mitochondrial ∆Ψm, indicative of apoptosis, can easily be detected using lipophilic, cationic fluorescent redistribution dyes such as ICT’s Fluorescent Mitochondrial Membrane Depolarization Assay reagents: tetramethylrhodamine ethyl ester (TMRE), tetramethylrhodamine methyl ester (TMRM), and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocarbocyanine iodide (JC-1). These dyes have a delocalized positive charge dispersed throughout their molecular structure, and yet their lipophilic solubility enables them to be readily membrane permeant and penetrate living cells. They redistribute across cell membranes according to the Nernst equation in a voltage-dependent manner. Accordingly, they possess a low membrane partition coefficient; meaning a low tendency to non-specifically associate with intracellular organelles and macro- molecules. These excellent potentiometric dyes also exhibit minimal self-quenching, low cytotoxicity, and are reasonably photostable. The Fluorescent Mitochondrial Membrane Depolarization Assay dyes exhibit very low toxicity and display rapid and reversible membrane equilibration properties. ICT’s JC-1 Fluorescent Mitochondrial Membrane Depolarization Assay kits easily distinguish between healthy, non-apoptotic cell populations and those cell populations that are transitioning into an apoptotic state. Inside a healthy, non-apoptotic cell, the lipophilic JC-1 dye, bearing a delocalized positive charge, enters the negatively charged mitochondria where it accumulates. JC-1 no longer accumulates inside the mitochondria, instead becoming more evenly distributed throughout the cytosol. When dispersed in this manner, overall cellular fluorescence levels drop dramatically. Healthy cells fluoresce orange and green, whereas cells with depolarized mitochondria fluoresce green and exhibit lower levels of orange fluorescence. JC-1 Fluorescent Mitochondrial Membrane Depolarization Assay kits can be used in conjunction with other existing research protocols. Grow cells following the usual cell cultivation protocol. If using an apoptosis induction model system, simply induce apoptosis according to the existing procedure, reserving a non-induced population of cells as a control. Once apoptosis has been induced in the cells, or the mitochondrial membrane has been depolarized by another method, such as using CCCP (included in the kit), spike JC-1 dye solution into each sample and control. Incubate the cells for 15-30 minutes at 37°C to allow JC-1 reagent to equilibrate within the polarized mitochondria. If the cells are not undergoing some form of metabolic or apoptotic stress, the mitochondrial ∆Ψm will remain intact, and JC-1 reagent will accumulate within the slightly negative/alkaline environment of the mitochondria and fluoresce brightly upon excitation. If the cells are apoptotic, the mitochondrial ∆Ψm will break down, causing JC-1 reagent to disperse throughout the cell cytosol. This results in a dramatic reduction in the fluorescence of the affected mitochondria, and as a result, overall cellular fluorescence is diminished significantly. Fluorescent Mitochondrial Membrane Depolarization Assays are for research use only. Not for use in diagnostic procedures.