JC-1

JC-1 (CBIC2) is an ideal fluorescent probe widely used to detect mitochondrial membrane potential. JC-1 accumulates in mitochondria in a potential dependent manner and can be used to detect the membrane potential of cells, tissues or purified mitochondria. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form a polymer, which emits strong red fluorescence (Ex=585 nm, Em=590 nm); When the mitochondrial membrane potential is low, JC-1 cannot aggregate in the matrix of mitochondria and produce green fluorescence (ex=510 nm, em= 527 nm)^[1]. In Vitro:Preparation of JC-1 staining solution:
1.1 Preparation of storage solution
Prepare a 5 mg/mL JC-1 solution using DMSO. Dissolve 5 mg of JC-1 in 1 mL of DMSO.
Note:
1) The storage solution of JC-1 is recommended to be aliquoted and stored at -20°C or -80°C in the dark.
1.2 Preparation of working solution (This method has been validated by MCE.)
Dilute the 5 mg/mL JC-1 storage solution in the following proportions: 1‰ DMSO + 89.9% ddH2O + 10% 10x PBS (HY-K3006). Prepare a 1-5 μg/mL JC-1 working solution. Take 1 μL of the 5 μg/mL working solution from the 5 mg/mL JC-1 storage solution and add it to a tube. Add 899 μL of ddH2O and mix well. Then, add 100 μL of 10x PBS to the above mixture and mix well.
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℃ 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.

JC-1 Staining:
a. 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℃.
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.
b. Transfer 0.5 mL of the cell suspension into a sterile centrifuge tube.
c. 400 g centrifugation for 3-5 min; Discard the supernatant.
d. The cells were resuspended with 1mljc-1 working solution and incubated in 5% CO₂ incubator at 37℃for 15-30 min.
e. Centrifugation at room temperature for 5 min at 400 g; Suck of the supernatant.
f. 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.
g. Resuspend the cells with 1mL of fresh culture medium or buffer, and immediately conduct subsequent flow cytometry or fluorescence microscope observation.
h. 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.