It is an ideal fluorescent probe for detecting mitochondrial membrane potential △Ψ m. When the membrane potential of mitochondria is high,JC-1 accumulates in the matrix of mitochondria and forms a polymer, which can produce red fluorescence. When the mitochondrial membrane potential is low,JC-1 cannot accumulate in the matrix of mitochondria, and at this time JC-1 is a monomer and can produce green fluorescence. The decline of mitochondrial membrane potential is a landmark event in the early stage of apoptosis. The decrease of cell membrane potential can be easily detected by the transformation of JC-1 from red fluorescence to green fluorescence, and the transformation of JC-1 from red fluorescence to green fluorescence can also be used as an early detection index of apoptosis.

Operation steps (for reference only) :

Positive control: CCCP (10mM) is recommended to be added to the cell culture medium at the ratio of 1:1000, diluted to 10μM, and treated for 20 minutes. Subsequently, JC-1 was loaded with the following method for the detection of mitochondrial membrane potential. For most cells, the membrane potential of mitochondria would be completely lost after 20 minutes of 10μM CCCP treatment, and JC-1 staining should be observed as green fluorescence. Normal cells stained with JC-1 should show red fluorescence. For specific cells, the concentration and time of action of CCCP may be different, which should be determined by referring to relevant literature.

Suspension cells:

(1) 100,000 to 600,000 cells were taken and re-suspended in 0.5mL cell culture medium, which could contain serum and phenol red.

(2) Add 0.5mL 1~5μg/mL JC-1 working liquid, reverse and mix it several times. The cells were incubated at 37 ℃ for 20 minutes.

(3) After incubation at 37℃, the cells were precipitated by centrifugation at 4 ℃ for 3 ~ 4 minutes at 600g. Discard the supernatant and take care not to remove the cells as much as possible.

(4) Wash with pre-cooled PBS or other appropriate solution twice: suspend the cells, centrifuge 600g at 4 ℃ for 3 ~ 4 minutes, precipitate the cells, discard the supernatant.

(5) After re-suspension with an appropriate amount of PBS or other appropriate solution, observation with fluorescence microscope or laser confocal microscope can also be detected by fluorescence spectrophotometer or flow cytometry.

Adherent cells:

Note: For adherent cells, if you want to use a fluorescence spectrophotometer or flow cytometry, you can collect the cells first and refer to the detection method of suspended cells after resuspension.

(1) For one hole of the six-well plate, remove the culture solution, wash the cells with PBS or other appropriate solution once according to the experimental requirements, and add 1mL of cell culture solution. The cell culture medium can contain serum and phenol red.

(2) Add 1mL of 1~5μg/mL JC-1 working liquid and mix thoroughly. The cells were incubated at 37℃ for 20 minutes.

(3) After incubation at 37℃, remove the supernatant and wash it twice with pre-cooled PBS or other appropriate solution.

(4) Add 2mL cell culture solution, which can contain serum and phenol red.

(5) Observation under fluorescence microscope or laser confocal microscope.

Purified mitochondria:

(1) 0.1mL purified mitochondria with a total protein content of 10-100μg were added into 0.9mL 0.2-1μg /mL JC-1 working solution.

(2) Detection with fluorescence spectrophotometer or fluorescence enzyme spectrometer: after mixing, time scan is performed directly with fluorescence spectrophotometer, excitation wavelength is 485nm, emission wavelength is 590nm. If you use a fluorometer, the excitation wavelength can not be set to 485nm when

The excitation wavelength is set in the range of 475 ~ 520nm.

(3) Observation with fluorescence microscope or laser confocal microscope.

Fluorescence observation and result analysis:

When detecting JC-1 monomer, the excitation light can be set to 490nm and the emission light can be set to 530nm. When detecting the JC-1 polymer, the excitation light can be set to 525nm and the emission light can be set to 590nm.

Note: It is not necessary to set the excitation and emission light at the maximum excitation wavelength and the maximum emission wavelength when determining fluorescence here. If using fluorescence microscopy, the detection of JC-1 monomer can refer to the setting of other green fluorescence observation, such as the setting of GFP or FITC; The detection of JC-1 polymers can refer to the Settings for observing other red fluorescence, such as propyl iodide or Cy3. The presence of green fluorescence indicates a decreased mitochondrial membrane potential, and the cell is likely in the early stages of apoptosis. The presence of red fluorescence indicates that the mitochondrial membrane potential is relatively normal, and the cell state is also relatively normal.

Note: After JC-1 probe is loaded and washed, the follow-up test should be completed within 30 minutes. Store in an ice bath before testing.

Examples of using this product（for reference only）

The normal human fetal hepatocyte cell line/L-02（10 μg/mL JC-1, 37℃; PBS）:

L-02 cells were cultured in 6-well plates (1×10⁵ cells/well) for 24 h, and then they were incubated with 200 μM H2O2 for 2 h. Then, the cells were separately incubated with the samples at different concentrations (2.5,5 μM) for 3 h in the dark at 37 ℃. ubsequently, the cells were stained by fresh JC-1 (10 μg/mL, 37 ℃). After that, the L-02 cells were collected and resuspended in PBS buffer. The fluorescence signals of cells were analyzed by flow cytometry (EX = 488 nm green signal, EM = 570?600 nm red signal).

Reference：

Ma H, Zhao J, Meng H, Hu D, Zhou Y, Zhang X, Wang C, Li J, Yuan J, Wei Y. Carnosine-Modified Fullerene as a Highly Enhanced ROS Scavenger for Mitigating Acute Oxidative Stress. ACS Appl Mater Interfaces. 2020 Apr 8;12(14):16104-16113. doi: 10.1021/acsami.0c01669. Epub 2020 Mar 24. PMID: 32186840.