JC-10 is an ideal fluorescent probe for detecting mitochondrial membrane potential △Ψm. Can detect cell, tissue or purified mitochondrial membrane potential. When the membrane potential of mitochondria is high, JC-10 accumulates in the matrix of mitochondria and forms a polymer, which can produce red fluorescence. When the mitochondrial membrane potential is low, JC-10 cannot accumulate in the matrix of mitochondria, and at this time JC-10 is a monomer and can produce green fluorescence. Thus, it is very convenient to detect changes in mitochondrial membrane potential through the transformation of fluorescence color. The proportion of mitochondrial depolarization is often measured by the relative ratio of red-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-10 from red fluorescence to green fluorescence, and the transformation of JC-10 from red fluorescence to green fluorescence can also be used as an early detection index of apoptosis.

The maximum excitation wavelength and emission wavelength of JC-10 monomer are 515nm and 529nm respectively. The maximum excitation wavelength of JC-10 polymer is 585nm and the maximum emission wavelength is 590nm. For actual observation, use the conventional Settings for observing red and green fluorescence.

Operation steps (for reference only) :

Setting of positive control:

10μM CCCP is recommended to treat cells for 20 minutes. Subsequently, JC-10 was loaded with the following method for the detection of mitochondrial membrane potential. For most cells, the membrane potential of mitochondria was completely lost after 20 minutes of 10μM CCCP treatment, and JC-10 staining showed green fluorescence. Normal cells should show red fluorescence after JC-10 staining. 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-10 dyeing solution, reverse and mix it several times. The cells were incubated at 37 ℃ for 20 minutes.

(3) During incubation, the buffer is placed in an ice bath and pre-cooled in advance.

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

(5) Wash with buffer twice: add 1mL buffer to resuspend cells, centrifuge 600g at 4 ℃ for 3 ~ 4 minutes, precipitate cells, discard supernatant. Then 1mL buffer was added to re-suspend the cells, 600g was centrifuged at 4℃ for 3 ~ 4 minutes, the cells were precipitated, and the supernatant was discarded.

(6) After re-suspension with an appropriate amount of buffer, observe with fluorescence microscope or laser confocal microscope, or detect with 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 once with PBS or other appropriate solution if necessary according to the specific experiment, and add 1mL of cell culture solution. The cell culture medium can contain serum and phenol red.

(2) Add 1mL 1~5μg/mL JC-10 dyeing solution and mix thoroughly. The cells were incubated at 37℃ for 20 minutes.

(3) During incubation, the buffer is placed in an ice bath and pre-cooled in advance.

(4) After incubation at 37℃, remove the supernatant and wash with buffer twice.

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

(6) 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-10 staining solution.

(2) Detection with fluorescence spectrophotometer or fluorescence enzyme spectrometer: after mixing, time scanning (timescan) is performed directly with fluorescence spectrophotometer, excitation wavelength is 485nm, emission wavelength is 590nm. If the fluorescence enzyme labeling instrument is used, when the excitation wavelength cannot be set to 485nm, the excitation wavelength can be set in the range of 475 ~ 520nm.

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

Fluorescence observation and result analysis:

When detecting JC-10 monomer, the excitation light can be set to 490nm and the emission light can be set to 530nm. When detecting JC-10 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-10 monomer can refer to the setting of other green fluorescence observation, such as the setting of GFP or FITC; The detection of JC-10 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:

1. The JC-10 reserve liquid will solidify and stick to the bottom, wall or cap of the centrifuge tube at low temperatures such as 4 ℃ and ice bath. It can be warmed in a water bath of 20 ~ 25℃ for a while until it is completely melted.

2. When washing with buffer after loading JC-10, keep the buffer about 4℃, and the washing effect is better at this time.

3. After JC-10 probe is loaded and washed, the follow-up test should be completed within 30 minutes as far as possible. Store in an ice bath before testing.

4. CCCP is a mitochondrial electron transport chain inhibitor, which is toxic, please pay attention to careful protection.

5. For your safety and health, please wear a lab coat and disposable gloves.