Product Introduction
The full English name of CCCP is Carbonyl Cyanide-m-Chlorophenylhydrazone. It is an ionophore and oxidative phosphorylation uncoupler, which can inhibit mitochondrial function, suppress transport processes, and retard cell growth. JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 accumulates in the mitochondrial matrix to form polymers, emitting red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot accumulate in the mitochondrial matrix and exists as monomers, emitting green fluorescence. The decrease in mitochondrial membrane potential can be easily detected by the fluorescence shift of JC-1 from red to green, which can also serve as an indicator for the early stage of cell apoptosis.
CCCP Solution (10 mmol/L) is a component of the Mitochondrial Membrane Potential Assay Kit (JC-1 Method). This kit uses JC-1 as the fluorescent probe to rapidly and sensitively detect changes in mitochondrial membrane potential in cells, tissues, or purified mitochondria, and can be applied to early cell apoptosis detection. CCCP serves as a positive control to induce the decrease of mitochondrial membrane potential. This reagent is for research use only and not intended for clinical diagnosis or any other purposes.
Materials to Be Prepared by Users
1. JC-1 Staining Solution, Phosphate Buffered Saline (PBS), Distilled Water or Deionized Water, Cell Culture Medium
2. Fluorescence Spectrophotometer or Flow Cytometer
Operating Procedures (For Reference Only)
1. Adding JC-1 Staining Solution: Add 1 mL of the solution to each well of a 6-well plate. Adjust the dosage appropriately for other culture vessels. For cell suspension, 0.5 mL of JC-1 Staining Solution is required for every 0.5-1.0×10⁶ cells.
2. Setting Positive Control: It is recommended to dilute the 10 mM CCCP solution in cell culture medium at a ratio of 1:1000 to a final concentration of 10 μM, and treat the cells for 20 min. Then load JC-1 according to the method described below to detect mitochondrial membrane potential. For most cell types, treatment with 10 μM CCCP for 20 min usually leads to a complete loss of mitochondrial membrane potential, and the cells should show green fluorescence after JC-1 staining. In contrast, normal cells should exhibit red fluorescence after JC-1 staining. For specific cell types, the effective concentration and treatment time of CCCP may vary, which should be determined by referring to relevant literature.
3. For Suspension Cells:
a. Take 1-6×10⁵ cells and resuspend them in 0.5 mL of cell culture medium, which may contain serum and phenol red.
b. Add 0.5 mL of JC-1 Staining Working Solution, invert the tube several times to mix well, and incubate the cells at 37℃ for 20 min in a cell incubator.
c. After incubation at 37℃, centrifuge the cells at 600×g and 4℃ for 3-4 min to pellet the cells, then discard the supernatant.
d. Wash the cells twice with PBS: Add 1 mL of PBS to resuspend the cells, centrifuge at 600×g and 4℃ for 3-4 min, discard the supernatant, and repeat this step once.
e. Resuspend the cells in a small amount of PBS, then observe them under a fluorescence microscope or laser confocal microscope. Detection can also be performed using a fluorescence spectrophotometer or analysis with a flow cytometer.
4. For Adherent Cells:
Note: For adherent cells, if detection by fluorescence spectrophotometer or flow cytometer is desired, the cells should be harvested first, resuspended, and then detected by referring to the method for suspension cells.
a. Aspirate the culture medium from the 6-well plate. If necessary according to the specific experiment, wash the cells once with PBS or other appropriate solution, then add 1 mL of cell culture medium, which may contain serum and phenol red.
b. Add 1 mL of JC-1 Staining Working Solution, mix thoroughly, and incubate the cells at 37℃ for 20 min in a cell incubator.
c. After incubation at 37℃, aspirate the supernatant and wash the cells twice with PBS.
d. Add 2 mL of cell culture medium, which may contain serum and phenol red.
e. Observe the cells under a fluorescence microscope or laser confocal microscope.
5. For Purified Mitochondria:
a. Further dilute the prepared JC-1 Staining Working Solution 5-fold.
b. Add 0.1 mL of purified mitochondria with a total protein amount of 10-100 μg to 0.9 mL of the diluted JC-1 Staining Working Solution.
c. Detection with Fluorescence Spectrophotometer or Fluorescence Microplate Reader: Mix well and directly perform time-scan detection using a fluorescence spectrophotometer with the excitation wavelength set at 485 nm and emission wavelength at 590 nm. If the fluorescence microplate reader cannot set the excitation wavelength to 485 nm, the excitation wavelength can be set within the range of 475-520 nm. Alternatively, the wavelength settings in Step 6 below can be referred to for fluorescence detection.
d. Observation with Fluorescence Microscope or Laser Confocal Microscope: Follow the method described in Step 6 below.
6. Fluorescence Observation and Result Analysis:
For detecting JC-1 monomers, set the excitation wavelength at 490 nm and emission wavelength at 530 nm. For detecting JC-1 polymers, set the excitation wavelength at 525 nm and emission wavelength at 590 nm. Note: It is not necessary to set the excitation and emission wavelengths at their maximum values for fluorescence measurement here. If using a fluorescence microscope for observation, the settings for detecting JC-1 monomers can refer to those used for observing other green fluorescent substances, such as GFP or FITC. The settings for detecting JC-1 polymers can refer to those used for observing other red fluorescent substances, such as Propidium Iodide (PI) or Cy3. The appearance of green fluorescence indicates a decrease in mitochondrial membrane potential, suggesting that the cells are likely in the early stage of apoptosis. The appearance of red fluorescence indicates that the mitochondrial membrane potential is relatively normal and the cell status is good.
Precautions
1. Avoid repeated freeze-thaw cycles of the JC-1 solution.
2. When washing the cells with PBS after JC-1 loading, keep the PBS at around 4℃ as much as possible to achieve a better washing effect.
3. Try to complete the subsequent detection within 30 min after JC-1 probe loading and washing. Store the samples on ice before detection.
4. CCCP is an inhibitor of the mitochondrial electron transport chain and has certain toxicity. Please handle it with care and take appropriate protective measures.
5. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
6. The volume of the reagent is small. Please centrifuge it at 12,000 rpm for 1 min before use.
7. For your safety and health, please wear a lab coat and disposable gloves during operation.
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