JC-1 (solution)

Guidelines (Following is our recommended protocol. This protocol only provides a guideline, and should be modified according to your specific needs).
1. Preparation of JC-1 working solution:
1‰ DMSO + 89.9% ddH₂O + 10% 10x PBS (HY-K3006). Prepare a 1.5-30 μM JC-1 working solution.
Note: 1) The JC-1 dye needs to be prepared immediately and used as soon as possible. It may precipitate if left for a period of time; try to prepare and use it under a light-proof condition.
2) At each step of the dilution process, ensure thorough mixing to achieve clarity. If necessary, ultrasonic assistance for dissolution at 37°C can be used.
3) Please do not directly dilute it with 1x PBS (HY-K3005) or serum-free medium to obtain the working solution; otherwise, it will lead to severe precipitation.
4) If the effect of JC-1 in entering the cells is not satisfactory, an appropriate amount of 20% Pluronic F127 solution can be added to the working solution, with a final concentration of 0.02-0.05%. Pluronic F127 can prevent JC-1 from aggregating in the buffer solution and help it enter the cells.
5) The storage solution of JC-1 is recommended to be aliquoted and stored at -20°C or -80°C in the dark.

2. JC-1 Staining:
2.1 Take the 6-well plate as an example for cell planking, and the density is 5×10⁵/mL. Incubate overnight in 5% CO₂ incubator at 37°C.
Note: It is suggested that the cell density during apoptosis induction should not exceed 1×10⁶/mL, which can also be cultured to the appropriate density according to your own cell type.
2.2 Transfer 0.5 mL of the cell suspension into a sterile centrifuge tube.
2.3 Take 0.5 mL suspension into sterile centrifuge tube; 400 g centrifugation for 3-5 min; Discard the supernatant.
2.4 The cells were resuspended with 1mL JC-1 working solution and incubated in 5% CO₂ incubator at 37°Cfor 15-30 min.
2.5 Centrifugation at room temperature for 5 min at 400 g; Suck of the supernatant.
2.6 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.
2.7 Resuspend the cells with 1mL of fresh culture medium or buffer, and immediately conduct subsequent flow cytometry or fluorescence microscope observation.
2.8 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.
Note: If used for enzyme labeling instrument, use 300 μL buffer resuspended cells; Then, transfer the stained cells to an opaque 96-well plate at a rate of 100 μL per well, and then conduct fluorescent enzyme label plate analysis.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only. Further protocols information, click here.