JC-1

1. Take the 6-well plate as an example for cell planking, and the density is 5×10^5/mL. Incubate overnight in 5% CO2 incubator at 37℃.
Note: it is suggested that the cell density during apoptosis induction should not exceed 1×10^6/ml, which can also be cultured to the appropriate density according to your own cell type.
2. Take 0.5 mL suspension into sterile centrifuge tube; 400 g centrifugation for 3-5 min; Discard the supernatant.
3. The cells were resuspended with 1mljc-1 working solution and incubated in 5% CO2 incubator at 37℃for 15-30 min.
4. Centrifugation at room temperature for 5 min at 400 g; Suck of the supernatant.
5. The cells were resuspended with 2 mL cell culture medium or buffer, and then centrifuged at room temperature for 5 min at 400 g; Discard the supernatant and repeat twice.
6. Resuspend the cells with 1mL of fresh culture medium or buffer, and immediately conduct subsequent flow cytometry or fluorescence microscope observation.
7. Data analysis (flow cytometry) : mitochondria of healthy cells containing red JC-1 aggregates were detected by FL2 channel; Apoptotic or unhealthy cells containing green JC-1 monomer were detected by FL1 (FITC) channel.
8. If used for enzyme labeling instrument, use 300 μL buffer resuspended cells; Then 100 per hole μ Transfer the stained cells to a light tight 96 well plate with the amount of L, and then conduct fluorescent enzyme label plate analysis.

JC-1 is dissolved in dimethylsulfoxide (DMSO) as a 2 mg/ml stock. JC-1 is excited at 490 nm and an optical image splitter device is attached to the microscope to separate spectrally the green and red components of JC-1 fluorescence.