Mitochondrial Membrane Potential Assay Kit (JC-1), IC-6569
Product Introduction
This product is a fluorescence-based assay kit for the qualitative and quantitative measurement of mitochondrial membrane potential (¦¤¦·m) in living cells using the cationic lipophilic dye JC-1 (5,5'',6,6''-tetrachloro-1,1'',3,3''-tetraethylbenzimidazolylcarbocyanine iodide). Mitochondrial membrane potential is a critical parameter of mitochondrial function and cellular health, generated by the proton gradient across the inner mitochondrial membrane during electron transport chain activity. In healthy, polarized mitochondria with high membrane potential, JC-1 spontaneously forms J-aggregates that emit red fluorescence (Ex/Em: 585/590 nm). In depolarized mitochondria with low membrane potential, JC-1 remains in its monomeric form, emitting green fluorescence (Ex/Em: 510/527 nm). This dual-emission property makes JC-1 a ratiometric indicator, with the ratio of red to green fluorescence providing a reliable measure of mitochondrial membrane potential that is independent of dye loading efficiency and mitochondrial mass. The assay is widely used for detecting mitochondrial depolarization during early apoptosis, evaluating drug-induced mitochondrial toxicity, and studying mitochondrial bioenergetics.
Product Features
1. Ratiometric Dual-Emission Probe: JC-1 forms red fluorescent J-aggregates in polarized mitochondria and green fluorescent monomers in depolarized mitochondria, enabling ratiometric (red/green) analysis that corrects for dye loading variability.
2. Early Apoptosis Detection: Mitochondrial depolarization is an early event in the intrinsic apoptotic pathway; JC-1 detects ¦¤¦·m loss preceding phosphatidylserine externalization and caspase activation.
3. Compatible with Multiple Detection Platforms: Suitable for analysis by flow cytometry, fluorescence microscopy, and fluorescence microplate reader.
4. Simple and Rapid Protocol: Single-step staining requiring only 20-30 minutes of incubation with no wash steps required before analysis for many applications.
5. Ready-to-Use Kit Format: Includes JC-1 dye, CCCP positive control, and optimized assay buffer for a complete mitochondrial membrane potential analysis workflow.
Specifications
Size: Available in 50 tests and 100 tests
Detection Method: Flow cytometry, fluorescence microscopy, or fluorescence microplate reader
Fluorophore: JC-1 (J-aggregates Ex/Em: 585/590 nm; Monomer Ex/Em: 510/527 nm)
Laser Lines: 488 nm (monomer and J-aggregate excitation)
Kit Components: JC-1 dye stock solution, CCCP (positive control), assay buffer
Sample Types: Suspension and adherent living cells
Storage and Stability
Storage Conditions: Store kit components at -20¡ãC, protected from light. JC-1 dye stock solution is light-sensitive and must be stored protected from light.
Shelf Life: The product is stable for 12 months from the date of manufacture when stored as directed. After reconstitution, the JC-1 working solution should be used immediately. Avoid repeated freeze-thaw cycles of the JC-1 stock solution.
Protocol (For Reference Only)
Important: JC-1 is light-sensitive. Protect dye and working solutions from light at all times. Prepare JC-1 working solution freshly immediately before use. CCCP positive control treatment should be performed to validate assay performance.
1. JC-1 Working Solution Preparation: Thaw the JC-1 stock solution at room temperature and protect from light. Dilute the JC-1 stock solution in pre-warmed assay buffer according to the kit instructions to prepare JC-1 working solution. Vortex briefly and centrifuge at 13,000 x g for 1-2 minutes to remove any insoluble particles. Use the supernatant for staining.
2. Positive Control Preparation: Treat an aliquot of cells with CCCP (carbonyl cyanide m-chlorophenyl hydrazone) at a final concentration of 10-50 µM for 15-30 minutes at 37¡ãC to induce mitochondrial depolarization. CCCP is a protonophore that collapses the mitochondrial membrane potential and serves as an essential positive control.
3. Cell Preparation: For suspension cells, collect 1-5¡Á10⁵ cells per sample by centrifugation. For adherent cells, harvest by gentle trypsinization, wash, and resuspend in assay buffer or culture medium. Include untreated cells as a negative control and CCCP-treated cells as a positive control.
4. JC-1 Staining: Resuspend cells in 500 µL of JC-1 working solution per sample. Incubate at 37¡ãC for 20-30 minutes in the dark. For flow cytometry, proceed directly to analysis without washing. For fluorescence microscopy, wash cells once with assay buffer to reduce background fluorescence before mounting.
5. Flow Cytometry Analysis: Analyze cells by flow cytometry using a 488 nm laser for excitation. Collect JC-1 monomer green fluorescence in the FITC/FL1 channel (525-530 nm) and J-aggregate red fluorescence in the PE/FL2 channel (585-590 nm). Create a bivariate scatter plot of red versus green fluorescence. Polarized cells with high ¦¤¦·m appear in the upper-left quadrant (high red, low green), while depolarized cells appear in the lower-right quadrant (low red, high green). Calculate the red/green fluorescence ratio for quantitative analysis.
6. Fluorescence Microscopy Analysis: After staining, wash cells once, resuspend in assay buffer, and mount on glass slides. Image using a fluorescence microscope equipped with FITC (green) and TRITC or Cy3 (red) filter sets. Polarized mitochondria appear red-orange, while depolarized mitochondria appear green.
Precautions
1. JC-1 is highly light-sensitive; protect dye stock and working solutions from light at all times. Prepare working solution immediately before use.
2. The JC-1 working solution should be pre-warmed to 37¡ãC and centrifuged to remove any undissolved dye aggregates before cell staining.
3. CCCP is a mitochondrial uncoupler and toxic; handle with appropriate safety precautions.
4. For flow cytometry, acquire data promptly after staining; prolonged incubation may lead to dye efflux from cells.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: How does JC-1 differ from other mitochondrial membrane potential dyes such as TMRE or TMRM?
A1: JC-1 provides ratiometric (red/green) detection that is independent of mitochondrial mass, dye loading, and photobleaching. TMRE and TMRM are single-emission dyes whose fluorescence intensity depends on both membrane potential and dye concentration. JC-1''s dual-emission property makes it more robust for quantitative comparisons across cell populations.
Q2: What is the purpose of the CCCP positive control?
A2: CCCP is a protonophore that collapses the mitochondrial proton gradient, causing complete mitochondrial depolarization. CCCP-treated cells serve as a positive control to define the depolarized (green monomer) gate and to validate that the assay is working properly.
Q3: Can I use this kit with fixed cells?
A3: No. JC-1 staining requires intact, living cells with functional mitochondrial membrane potential. Fixation permeabilizes the mitochondrial membrane, abolishing the membrane potential and rendering JC-1 incapable of forming J-aggregates.
Q4: What is the expected red/green ratio change during apoptosis?
A4: In healthy polarized cells, the red/green fluorescence ratio is typically high (J-aggregate dominant). During early apoptosis, mitochondrial depolarization causes a decrease in the red/green ratio as J-aggregates dissociate and green monomer fluorescence increases. The degree of change depends on the apoptotic stimulus and cell type.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of staining conditions is recommended for specific cell types and experimental systems.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product. CCCP is a mitochondrial uncoupler and toxic; review the Safety Data Sheet before handling.
Ordering Information
Catalog Number: IC-6569
Product Name: Mitochondrial Membrane Potential Assay Kit (JC-1)
Size: 50 tests / 100 tests
Price:
50 tests: CNY ¥1500.00 / USD $150.00 / EUR €180.00 / JPY ¥27000.00
100 tests: CNY ¥2100.00 / USD $210.00 / EUR €252.00 / JPY ¥37800.00
2023 Version
