Mitochondrial Membrane Potential Assay Kit (JC-1)
Dual-fluorescence ratio measurements yield more reliable results.
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Instruction manual- Product Description
- Composition
- Precautions
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Product Overview
The Mitochondrial Membrane Potential Assay Kit with JC‑1 is a reagent kit that uses JC‑1 as a fluorescent probe to rapidly and sensitively measure changes in mitochondrial membrane potential in cells, tissues, or purified mitochondria, making it suitable for early detection of apoptosis. JC‑1 is an ideal fluorescent probe widely employed to assess mitochondrial membrane potential (ΔΨm). It can be used to monitor the membrane potential of intact cells, tissue samples, or isolated mitochondria. When mitochondrial membrane potential is high, JC‑1 aggregates within the mitochondrial matrix, forming J‑aggregates that emit red fluorescence; conversely, at lower membrane potentials, JC‑1 remains monomeric and emits green fluorescence. Thus, changes in mitochondrial membrane potential can be detected by monitoring the shift in fluorescence color. The relative ratio of red-to-green fluorescence is commonly used to quantify the extent of mitochondrial depolarization. A decline in mitochondrial membrane potential is an early hallmark of apoptosis. The transition from red to green fluorescence observed with JC‑1 provides a straightforward means of detecting mitochondrial membrane potential loss and can serve as an early biomarker for apoptosis.
This kit provides CCCP as a positive control to induce a decrease in mitochondrial membrane potential. For samples cultured in six-well plates, the kit can analyze up to 30 samples; for samples cultured in twelve-well plates, it can analyze up to 60 samples.
Storage conditions
Ice pack transport. Store at −20°C away from light; for JC‑1 (200×), minimize repeated freeze–thaw cycles; shelf life: one year.
Detection method
1. Setting of the positive control:
It is recommended to add the CCCP (50 mM) provided in the kit to the cells at a 1:1,000 dilution, resulting in a final concentration of 50 μM, and to treat the cells for 10–20 minutes. Subsequently, load JC‑1 according to the procedure described below to assess mitochondrial membrane potential. For most cell types, a 50 μM CCCP treatment for 10–20 minutes typically leads to complete loss of mitochondrial membrane potential; after JC‑1 staining, the fluorescence should appear green. By contrast, healthy cells stained with JC‑1 should exhibit red fluorescence. For certain cell types, the effective concentration and incubation time of CCCP may vary, so it is necessary to optimize these parameters empirically.
2. Preparation of JC-1 staining working solution:
For a 6-well plate, each well requires 1 mL of JC‑1 staining working solution; for a 12-well plate, each well requires 0.5 mL. The volume of JC‑1 staining working solution for other culture vessels should be scaled accordingly. For cell suspensions, 0.5 mL of JC‑1 staining working solution is needed per 500,000–1,000,000 cells. Refer to the table below for the preparation of JC‑1 staining working solution:
Total volume 1 mL 10 mL JC-1 (200×) 10 µL 100 µL Ultrapure water 790 µL 7.9 mL JC-1 Staining Buffer (5×) 200 µL 2 mL ▲JC‑1 (200×), first dilute with ultrapure water according to the specified ratio and vigorously shake to ensure complete dissolution, then add 5× staining buffer; after mixing, you will obtain the JC‑1 staining working solution.
3. For suspension cells:
1) Collect 100,000 to 500,000 cells and resuspend them in 0.5 mL of cell culture medium; the medium may contain serum and phenol red.
2) Add 0.5 mL of JC-1 staining working solution and gently invert several times to mix. Incubate in a cell culture incubator at 37°C for 15–30 minutes.
3) During the incubation period, prepare an appropriate volume of 1× JC-1 staining buffer by adding 4 mL of distilled water to every 1 mL of 5× JC-1 staining buffer, and keep it on ice.
4) After incubation at 37°C, centrifuge at 1500 rpm at 4°C for 3–4 minutes, and discard the supernatant.
5) Wash the cells 1–2 times with JC-1 staining buffer (1×).
6) Resuspend the cells in an appropriate volume of JC‑1 staining buffer (1×), then observe them using a fluorescence microscope or a laser confocal microscope. Alternatively, you may perform fluorescence spectrophotometric measurements or analyze them with a flow cytometer.
4. For adherent cells:
Note: For adherent cells, if you wish to perform detection using a fluorescence spectrophotometer or a flow cytometer, you can first collect the cells, resuspend them, and then follow the detection protocol for suspension‑type cells.
1) For a single well of a six-well plate, aspirate the culture medium. If necessary, wash the cells once with PBS or another appropriate solution, as specified by the experimental protocol, then add 1 mL of cell culture medium. The culture medium may contain serum and phenol red.
2) Add 1 mL of JC-1 staining working solution and mix thoroughly. Incubate in a cell culture incubator at 37°C for 15–30 minutes.
3) During the incubation period, prepare an appropriate volume of 1× JC-1 staining buffer by adding 4 mL of distilled water to every 1 mL of 5× JC-1 staining buffer, and keep it on ice.
4) After incubation at 37°C, aspirate the supernatant and wash twice with JC-1 staining buffer (1×).
5) Add 2 mL of JC-1 staining buffer (1×), and observe under a fluorescence microscope or a laser confocal microscope.
5. For purified mitochondria:
1) Further dilute the prepared JC-1 staining working solution fivefold with 1× JC-1 staining buffer.
2) Add 0.1 mL of purified mitochondria, with a total protein content of 10–100 µg, to 0.9 mL of a 5-fold diluted JC‑1 staining working solution.
3) Detection using a fluorescence spectrophotometer or a fluorescence microplate reader: After mixing, perform a time‑scan directly with a fluorescence spectrophotometer, setting the excitation wavelength to 485 nm and the emission wavelength to 590 nm. If using a fluorescence microplate reader and the excitation wavelength cannot be set to 485 nm, choose an excitation wavelength within the range of 475–520 nm. Alternatively, you may also follow the wavelength settings described in Step 6 below for fluorescence detection.
4) Observe using a fluorescence microscope or a laser confocal microscope: the method is the same as in step 6 below.
6. Fluorescence Observation and Result Analysis:
1) When detecting JC-1 monomers, the excitation wavelength can be set to 490 nm and the emission wavelength to 530 nm; when detecting JC-1 aggregates, the excitation wavelength can be set to
525 nm, with the emission wavelength set to 590 nm. ▲When measuring fluorescence here, it is not necessary to align the excitation and emission wavelengths with their respective maximum values.
When observing with a fluorescence microscope, use settings similar to those for other green fluorophores—such as GFP or FITC—when detecting JC‑1 monomers; for JC‑1 aggregates, refer to the settings used for other red fluorophores, such as propidium iodide or Cy3. The presence of green fluorescence indicates a depolarized mitochondrial membrane potential, suggesting that the cell is likely in the early stages of apoptosis. Red fluorescence, on the other hand, suggests a relatively normal mitochondrial membrane potential and a generally healthy cellular state.
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Product ingredients
Component Name Packaging Numbering JC-1 (200x) 200 µL × 5 tubes 100-117A CCCP (50 mM) 30 µL 100-117B Ultrapure water 90 mL 100-117C -
Precautions
1. JC-1 (200×) and CCCP should be allowed to equilibrate at room temperature (20–25°C) before use.
2. JC‑1 (200×) must first be thoroughly dissolved and mixed with the ultrapure water provided in the kit before adding the JC‑1 staining buffer (5×). Do not prepare the JC‑1 staining buffer (1×) first and then add JC‑1 (200×); this will make it difficult for JC‑1 to dissolve completely, severely compromising subsequent assays.
3. After loading the JC‑1, wash with 1× JC‑1 staining buffer, maintaining the buffer at approximately 4°C; this temperature yields optimal washing results.
4. After loading and washing with the JC‑1 probe, complete the subsequent analysis within 30 minutes whenever possible. Prior to analysis, store the sample on ice.
5. Do not dilute the JC‑1 staining buffer (5×) completely to prepare a 1× JC‑1 staining buffer; during use, this kit requires direct application of the JC‑1 staining buffer (5×).
6. If precipitation is observed in the JC‑1 staining buffer (5×), it must be completely dissolved before use; to facilitate dissolution, the solution may be heated at 37°C.
7. CCCP is an inhibitor of the mitochondrial electron transport chain and is harmful to humans; please take appropriate protective measures when using it.
8. For your safety and health, please wear a lab coat and disposable gloves while performing this procedure.
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