Mitochondria play a pivotal role in the process of apoptosis. A variety of apoptosis stimulating factors can induce apoptosis in different cells, and the decrease of mitochondrial transmembrane potential is considered to be the earliest in the process of apoptosis cascade. The event that occurs before the appearance of nuclear apoptotic features (chromatin condensation, DNA fragmentation) occurs, and once mitochondrial DYmt collapses, apoptosis is irreversible.
The presence of mitochondrial transmembrane potentials makes some lipophilic cationic fluorescent dyes such as:
1.Rhodamine 123
2.3,3-Dihexyloxacarbocyanine iodide[DiOC6(3)]
3.Tetrechloro-tetraethylbenzimidazol carbocyanine iodide[JC-1]
4.Tetramethyl rhodamine methyl ester (TMRM)
It can be bound to the mitochondrial matrix, and the increase or decrease in fluorescence indicates an increase or decrease in the electronegativity of the mitochondrial inner membrane.
method
Normally cultured cells and apoptosis-inducing cells were added to a final concentration of Rhodamine 123 (1 mM) or a final concentration of DiOC6 (25 nM), JC-1 (1 mM), TMRM (100 nM), equilibrated at 37 ° C for 30 min, flow The cytometer measures the fluorescence intensity of the cells.
Precautions
1. Always maintain a consistent pH value in the dye solution, as changes in pH will affect the membrane potential.
2. If the protein suspension is equilibrated with the dye, they will bind to some of the dyes, reducing the concentration of the dye and causing false depolarization.
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