1. Preparation of JC-1 dyeing working solution
The amount of JC-1 staining working solution required for each well of the six-well plate is 1 mL, and the amount of JC-1 staining working solution for other culture vessels is similar: 0.5 mL of JC-1 staining working solution is required for every 500,000 to 1 million cells in the cell suspension. Dilute an appropriate amount of JC-1 (200 ×) by adding 8 mL of ultrapure water for every 50 μL of JC-1 (200 ×). Thoroughly dissolve and mix JC-1 with vigorous shaking. Then add 2 mL of JC-1 staining buffer (5 ×), and mix well to obtain JC-1 staining working solution.

2. Setting of positive control:
CCCP (10 mM) provided in the kit is recommended to be added to the cell culture medium at a ratio of 1: 1000, diluted to 10 μM, and treated the cells for 20 minutes. Subsequently, JC-1 was loaded according to the following method, and mitochondrial membrane potential was detected. For most cells, the membrane potential of mitochondria will usually be completely lost after 20 minutes of 10 μM CCCP treatment, and it should show green fluorescence after JC-1 staining; However, normal cells should show red fluorescence after staining with JC-1. For specific cells, the concentration and duration of CCCP may be different, and it is necessary to refer to relevant literature to decide.

3. For suspension cells
Take 100,000 to 600,000 cells and resuspend them in 0.5 mL cell culture medium. The cell culture medium can contain serum and phenol red.
Add 0.5 mL of JC-1 staining working solution, reverse it several times and mix well. Incubate in a cell incubator at 37 °C for 20 minutes.
During the incubation period, an appropriate amount of JC-1 staining buffer (1 ×) was prepared at a ratio of 4 mL of distilled water per 1 mL of JC-1 staining buffer (5 ×) and placed in an ice bath.
After the incubation at 37 °C, 600g was centrifuged at 4 °C for 3-4 minutes to pellet the cells. Discard the supernatant and be careful not to aspirate the cells as much as possible.
Wash twice with JC-1 staining buffer (1 ×): add 1 mL of JC-1 staining buffer (1 ×) to resuspend the cells, centrifuge at 600 g at 4 °C for 3-4 minutes, pellet the cells, and discard the supernatant.
Add 1mL JC-1 staining buffer (1 ×) to resuspend the cells, centrifuge at 600g at 4 °C for 3-4 minutes, pellet the cells, and discard the supernatant. After resuspending with an appropriate amount of JC-1 staining buffer (1 ×), observe with a fluorescence microscope or laser confocal microscope, or detect with a fluorescence spectrophotometer or analyze with a flow cytometer.

4. For adherent cells
Note: For adherent cells, if you want to use fluorescence spectrophotometer or flow cytometer detection, you can collect the cells first, and refer to the detection method of suspended cells after resuspension.
For one well of the six-well plate, the broth is aspirated, and the cells can be washed once with PBS or other appropriate solution if necessary depending on the specific experiment, and 1 mL of the cell broth is added. The cell culture medium may contain serum and phenol red. Add 1 mL of JC-1 staining working solution and mix well. Incubate in a cell incubator at 37 °C for 20 minutes.
During the incubation period, an appropriate amount of JC-1 staining buffer (1 ×) was prepared at a ratio of 4 mL of distilled water per 1 mL of JC-1 staining buffer (5 ×) and placed in an ice bath.
After the incubation at 37 °C was completed, the supernatant was aspirated and washed twice with JC-1 staining buffer (1 ×).
2 mL of cell culture medium, which may contain serum and phenol red, is added.
Observe under fluorescence microscope or laser confocal microscope.

5. For purified mitochondria
The prepared JC-1 staining working solution was diluted 5 times with JC-1 staining buffer (1 ×).
0.9 mL of 5-fold diluted JC-1 staining working solution was added with 0.1 mL of purified mitochondria with a total protein content of 10-100 μg. Detection with a fluorescence spectrophotometer or a fluorescence microplate reader: After mixing, directly use a fluorescence spectrophotometer to time scan (timescan). The excitation wavelength is 485nm and the emission wavelength is 590nm. If a fluorescent microplate reader is used and the excitation wavelength cannot be set to 485 nm, the excitation wavelength can be set in the range of 475 to 520 nm. In addition, fluorescence detection can also be performed with reference to the wavelength setting in step 6 below.
Observation with fluorescence microscope or laser confocal microscope: The method is the same as step 6 below.

6. Fluorescence observation and result analysis and detection
In the case of JC-1 monomer, the excitation light can be set to 490 nm and the emission light can be set to 530 nm; When the JC-1 polymer is detected, the excitation light can be set to 525 nm and the emission light can be set to 590 nm. Note that it is not necessary to set the excitation light and the emission light at the maximum excitation wavelength and the maximum emission wavelength when measuring fluorescence here. For observation using a fluorescence microscope, when detecting JC-1 monomer, you can refer to the settings when observing other green fluorescence, such as the settings when observing GFP or FITC; When detecting JC-1 polymer, you can refer to the settings when observing other red fluorescence, such as propidium iodide or Cy3. The appearance of green fluorescence indicates that the mitochondrial membrane potential decreases, and the cell is probably in the early stage of apoptosis. The appearance of red fluorescence indicates that the mitochondrial membrane potential is relatively normal and the state of cells is also relatively normal.