1. Preparation of JC-1 staining solution

1.1 Prepare JC-1 Stock Solution. The stock solution should be prepared in dimethyl sulfoxide (DMSO, concentration 5 mg/mL. Following preparation, the stock solution should be aliquoted and stored in a -20°C freezer. It is important to avoid repeated freeze/thaw cycles. It is important to avoid repeated freeze/thaw cycles. The solution can be stored for up to 6 months.)

1.2 Preparation of Working Solution

Please prepare 1-20 μg/mL of JC-1 working solution by diluting the stock solution with pre-warmed serum-free cell culture medium or PBS.

Please note that the final concentration of the working solution is dependent on the experimental system. It is therefore recommended that the final concentration conditions be optimised.

2. Cell Staining Note:

If JC-1 does not enter the cells effectively, add a 20% Pluronic F127 solution to the working solution at a final concentration of 0.02-0.05%. Pluronic F127 prevents JC-1 from polymerising in the buffer and facilitates its entry into the cells.

2.1 The cells should be spread in a 6-well plate at a density of 5 x 10^5 cells/mL. They should then be incubated overnight at 37 °C in a 5% CO2 incubator.

2.2. A quantity of 0.5 mL of cell suspension was transferred into a sterile centrifuge tube.

2.3. Centrifuge at 400 g for 3-5 minutes, then discard the fluid.

2.4 The cells should be resuspended in 1 mL of JC-1 working solution, after which they should be incubated for 15-30 minutes at 37°C in a 5% CO2 incubator.

2.5. Centrifuge at 400 g for 5 minutes at room temperature, then carefully aspirate the upper layer.

2.6. The cells should be resuspended in 2 mL of cell culture medium or buffer, and then centrifuge at 400 g for 5 minutes at room temperature. The upper layer should be discarded, and this step should be repeated twice.

2.7. The cells should be resuspended in 1 mL of fresh culture medium or buffer, and the flow analysis or fluorescence microscopy should be performed immediately.

2.8 Data analysis (flow cytometry): healthy cells containing red JC-1 aggregates are detected by the FL2 channel, while apoptotic or unhealthy cells containing green JC-1 monomers are detected by the FL1 (FITC) channel.

Please note: When performing an ELISA, the cells must be resuspended in 300 μL of buffer and then transferred.