This plan only provides a guideline. Please modify it according to your specific needs.
1. Prepare the JC-1 staining working solution
(1) This product is provided in the form of a 5mM DMSO stock solution. At low temperatures, it will solidify and stick to the bottom, walls or cover of the centrifuge tube. It can be incubated in a water bath at 20-25°C for a short period until it fully melts before use. It is recommended to centrifuge the product before first use, then aliquot and store at -20°C or -80°C protected from light.
(2) Prepare a JC-1 working solution with a concentration of 1-10μM: Take 5μM as an example. Add 1μL of JC-1 stock solution (5mM) to 800μL of sterile water and shake vigorously until thoroughly dissolved and evenly mixed. Then add 200μL of JC-1 staining buffer (5×) and mix well to obtain the JC-1 staining working solution.
Note: The JC-1 dye is difficult to dissolve. It must be dissolved in sterile water first, and ultrasonicated for 3-5 minutes until completely dissolved and mixed. Then add the JC-1 staining buffer (5×).
2. Set up a positive control
The CCCP (20mM) provided in the kit is recommended to be added to the staining working solution at a ratio of 1:1000, diluted to 20μM.
For most cells, the mitochondrial membrane potential will completely disappear after 20 minutes of treatment with 20μM CCCP, but factors such as cell density, drug resistance, and metabolic level will affect the sensitivity of cells to CCCP treatment. When using for the first time, it is recommended to conduct gradient exploration of the CCCP concentration, with the gradient range referring to 10-30μM.
3. Staining of suspended cells
(1) The recommended cell density for most cell lines is 0.2-1.2×10⁶ cells/ml. The optimal cell density for each specific cell line needs to be determined separately. Take 1mL of cell suspension, centrifuge at 600g for 3-5 minutes. Discard the supernatant and wash once with PBS.
(2) Add 1mL of JC-1 staining working solution and invert several times to mix well. Incubate in the cell culture box at 37°C for 20 minutes.
(3) During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1×) at a ratio of 4mL of sterile water for every 1mL of JC-1 staining buffer (5×), and place it on an ice bath.
(4) After incubation at 37°C, centrifuge at 600g, 4°C for 3-5 minutes, and precipitate the cells. Discard the supernatant, and try to avoid absorbing the cells as much as possible.
(5) Wash twice with JC-1 staining buffer (1×) or cell culture medium (which can contain serum and phenol red). Resuspend the cells with 1mL of JC-1 staining buffer (1×), centrifuge at 600g, 4°C for 3-5 minutes, precipitate the cells, and discard the supernatant. Then resuspend the cells with 1mL of JC-1 staining buffer (1×), centrifuge at 600g, 4°C for 3-5 minutes, precipitate the cells, and discard the supernatant.
(6) Take an appropriate amount of JC-1 staining buffer (1×) or cell culture medium to resuspend the cells, and observe with a fluorescence microscope or confocal laser scanning microscope. Alternatively, detect with a fluorescence spectrophotometer or analyze with a flow cytometer.
4. Staining of adherent cells (for 6-well plates as an example)
Note: For adherent cells, if using a fluorescence spectrophotometer or flow cytometer, collect and resuspend the cells first, then follow the procedure for suspension cells. JC-1 detection is recommended at 60-70% confluence.
(1) Remove the culture medium from the 6-well plate and wash the cells once with PBS or other appropriate solution.
(2) Add 2mL of JC-1 staining working solution and mix well. Incubate in the cell culture box at 37°C for 20 minutes.
(3) During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1×) by adding 4mL of sterile water to 1mL of JC-1 staining buffer (5×), and store it in an ice bath.
(4) After incubation at 37°C, remove the supernatant and wash twice with JC-1 staining buffer (1×).
(5) Add an appropriate amount of JC-1 staining buffer (1×) or cell culture medium for cell culture, and observe with a fluorescence microscope or laser confocal microscope.
5. Purification of Mitochondria Staining
(1) Dilute the prepared JC-1 staining working solution with JC-1 staining buffer (1×) by a factor of 5.
(2) Add 0.1mL of the 5-fold diluted JC-1 staining working solution to 0.9mL of total protein with a quantity ranging from 10 to 100µg of purified mitochondria.
(3) Mix well and immediately perform a time scan using a fluorescence spectrophotometer or a fluorescence microplate reader.
Note: If observing with a fluorescence microscope or a confocal laser scanning microscope, incubate at 37°C for 10-15 minutes, wash, and then load onto the instrument.
6. Fluorescence observation and result analysis
When detecting JC-1 monomers, set the excitation light to 490nm and the emission light to 530nm (green fluorescence); when detecting JC-1 polymers, set the excitation light to 525nm and the emission light to 590nm (red fluorescence).
Note: When measuring fluorescence, it is not necessary to set the excitation light and emission light to the maximum excitation wavelength and maximum emission wavelength. If using a fluorescence microscope for observation, when detecting JC-1 monomers, you can refer to the settings used when observing other green fluorescence, such as GFP or FITC; when detecting JC-1 polymers, you can refer to the settings used when observing other red fluorescence, such as propidium iodide or Cy3.
7. Precautions
(1) Use live cells in the logarithmic growth phase to ensure good cell condition.
(2) After loading JC-1, when washing with JC-1 staining buffer (1×), keep the JC-1 staining buffer (1×) at 4°C or slightly lower. At this time, the washing effect is better. Complete the subsequent detection as soon as possible within 30 minutes after washing. Store in an ice bath before detection.
(3) Fluorescent dyes all have quenching problems. Please try to avoid light exposure to slow down fluorescence quenching. The red JC-1 fluorescence decays faster than the green fluorescence. It is recommended to take images of the stained cells in the red channel first.
(4) For your safety and health, please wear a lab coat and use disposable gloves during operation.
