JC-1 Fluorescent Mitochondrial Membrane Depolarization Assay

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JC-1 accumulates and fluoresces orange in polarized mitochondria. Following membrane depolarization, JC-1 disperses within the cell and fluoresces green. Analyze using a flow cytometer, fluorescence plate reader, or fluorescence microscopy.

Figure 1. Jurkat cells were treated with DMSO (negative control) or 1 µM staurosporine for 2 hours to induce apoptosis. Cells were then stained with JC-1 dye. Normal healthy cells (two cells, upper and lower left) concentrate JC-1 in mitochondria and fluoresce bright orange. Apoptotic cells (three cells, right), with mitochondria of various stages of permeability, exhibit a reduced orange fluorescence and increased green fluorescence, as JC-1 disperses through the cells. (Dr. Brian Lee, ICT).
Figure 2. Jurkat and HL-60 cells were exposed to DMSO (negative control) (dark orange) or 50 μM CCCP depolarizing agent (light orange) and then incubated with JC-1 for 20 minutes at 37°C, and washed. Aliquots were analyzed in triplicate in a fluorescence plate reader set at 488 nm excitation and 590 nm emission filter settings. Healthy cells exhibited a high level of orange fluorescence; the CCCP-depolarized cells had reduced orange fluorescence as the dye dispersed. (Ms. Tracy Hanson, ICT)
Figure 3. Jurkat cells were treated with 1 µM staurosporine, an apoptosis-inducing agent (black), or DMSO (negative control) (red), stained with JC-1, and analyzed by flow cytometer. Apoptotic cells (black) exhibit much less fluorescence compared to healthy cells (right) which concentrate JC-1 in their polarized mitochondria. Almost all cells treated with staurosporine exhibit apoptosis-associated mitochondrial depolarization (Ms. Tracy Hanson, ICT, 13A54, 050113).
Figure 4. Jurkat cells were treated with either DMSO (Negative Control) or staurosporine (Positive Control) for 3 hours at 37°C and labeled with JC-1 for 15 minutes. Cells were analyzed with a FACS Caliber Becton Dickinson Flow Cytometer. Collapse of the mitochondrial membrane potential gradient is indicated by an increase in the number of cells falling from P2 (Green) into P3 (Pink) following induction of apoptosis (Dr. Brian Lee, ICT).
Figure 1. Jurkat cells were treated with DMSO (negative control) or 1 µM staurosporine for 2 hours to induce apoptosis. Cells were then stained with JC-1 dye. Normal healthy cells (two cells, upper and lower left) concentrate JC-1 in mitochondria and fluoresce bright orange. Apoptotic cells (three cells, right), with mitochondria of various stages of permeability, exhibit a reduced orange fluorescence and increased green fluorescence, as JC-1 disperses through the cells. (Dr. Brian Lee, ICT).
Figure 1. Jurkat cells were treated with DMSO (negative control) or 1 µM staurosporine for 2 hours to induce apoptosis. Cells were then stained with JC-1 dye. Normal healthy cells (two cells, upper and lower left) concentrate JC-1 in mitochondria and fluoresce bright orange. Apoptotic cells (three cells, right), with mitochondria of various stages of permeability, exhibit a reduced orange fluorescence and increased green fluorescence, as JC-1 disperses through the cells. (Dr. Brian Lee, ICT).
Figure 2. Jurkat and HL-60 cells were exposed to DMSO (negative control) (dark orange) or 50 μM CCCP depolarizing agent (light orange) and then incubated with JC-1 for 20 minutes at 37°C, and washed. Aliquots were analyzed in triplicate in a fluorescence plate reader set at 488 nm excitation and 590 nm emission filter settings. Healthy cells exhibited a high level of orange fluorescence; the CCCP-depolarized cells had reduced orange fluorescence as the dye dispersed. (Ms. Tracy Hanson, ICT)
Figure 3. Jurkat cells were treated with 1 µM staurosporine, an apoptosis-inducing agent (black), or DMSO (negative control) (red), stained with JC-1, and analyzed by flow cytometer. Apoptotic cells (black) exhibit much less fluorescence compared to healthy cells (right) which concentrate JC-1 in their polarized mitochondria. Almost all cells treated with staurosporine exhibit apoptosis-associated mitochondrial depolarization (Ms. Tracy Hanson, ICT, 13A54, 050113).
Figure 4. Jurkat cells were treated with either DMSO (Negative Control) or staurosporine (Positive Control) for 3 hours at 37°C and labeled with JC-1 for 15 minutes. Cells were analyzed with a FACS Caliber Becton Dickinson Flow Cytometer. Collapse of the mitochondrial membrane potential gradient is indicated by an increase in the number of cells falling from P2 (Green) into P3 (Pink) following induction of apoptosis (Dr. Brian Lee, ICT).

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Product Manual
Safety Data Sheet

SKU: 911

Size: 400 Tests
1-2 business days
Price:
$495.00

Mitochondria play a central role in the biochemical processes associated with the life and death stages of eukaryotic cells. Under normal physiological conditions, a membrane-based proton pump generates an electrochemical gradient, enabling the production of ATP to drive cellular energy-dependent processes. The oxidation of glucose and fatty acids by enzymes associated with the mitochondrial respiratory chain establishes a proton and pH gradient across the mitochondrial inner membrane, resulting in a transmembrane electrical potential gradient (∆Ψm) of -80 to -120 mV and a pH gradient of 0.5-1.0 pH units.

Depolarization of the inner mitochondrial membrane can lead to an opening of the mitochondrial permeability transition pore (PTP). This results in the leakage of intermembrane proteins, including cytochrome C, that facilitate the induction of apoptosis through apoptosome formation. Caspase activation has been shown to accelerate the process of ∆Ψm loss. Moreover, a feedback mechanism that results in the generation of reactive oxygen species further accelerates the rate of cell death. Because mitochondrial dysfunction has been closely tied to such neurodegenerative diseases as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis, mitochondria remain an important organelle of study.

Loss of mitochondrial ∆Ψm, indicative of apoptosis, can easily be detected using lipophilic, cationic fluorescent redistribution dyes such as ICT’s Fluorescent Mitochondrial Membrane Depolarization Assay reagents: tetramethylrhodamine ethyl ester (TMRE), tetramethylrhodamine methyl ester (TMRM), and 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolocarbocyanine iodide (JC-1). These dyes have a delocalized positive charge dispersed throughout their molecular structure, and yet their lipophilic solubility enables them to be readily membrane permeant and penetrate living cells. They redistribute across cell membranes according to the Nernst equation in a voltage-dependent manner. Accordingly, they possess a low membrane partition coefficient; meaning a low tendency to non-specifically associate with intracellular organelles and macro- molecules. These excellent potentiometric dyes also exhibit minimal self-quenching, low cytotoxicity, and are reasonably photostable. The Fluorescent Mitochondrial Membrane Depolarization Assay dyes exhibit very low toxicity and display rapid and reversible membrane equilibration properties.

ICT’s JC-1 Fluorescent Mitochondrial Membrane Depolarization Assay kits easily distinguish between healthy, non-apoptotic cell populations and those cell populations that are transitioning into an apoptotic state. Inside a healthy, non-apoptotic cell, the lipophilic JC-1 dye, bearing a delocalized positive charge, enters the negatively charged mitochondria where it accumulates. JC-1 no longer accumulates inside the mitochondria, instead becoming more evenly distributed throughout the cytosol. When dispersed in this manner, overall cellular fluorescence levels drop dramatically. Healthy cells fluoresce orange and green, whereas cells with depolarized mitochondria fluoresce green and exhibit lower levels of orange fluorescence.

JC-1 Fluorescent Mitochondrial Membrane Depolarization Assay kits can be used in conjunction with other existing research protocols. Grow cells following the usual cell cultivation protocol. If using an apoptosis induction model system, simply induce apoptosis according to the existing procedure, reserving a non-induced population of cells as a control. Once apoptosis has been induced in the cells, or the mitochondrial membrane has been depolarized by another method, such as using CCCP (included in the kit), spike JC-1 dye solution into each sample and control. Incubate the cells for 15-30 minutes at 37°C to allow JC-1 reagent to equilibrate within the polarized mitochondria. If the cells are not undergoing some form of metabolic or apoptotic stress, the mitochondrial ∆Ψm will remain intact, and JC-1 reagent will accumulate within the slightly negative/alkaline environment of the mitochondria and fluoresce brightly upon excitation. If the cells are apoptotic, the mitochondrial ∆Ψm will break down, causing JC-1 reagent to disperse throughout the cell cytosol. This results in a dramatic reduction in the fluorescence of the affected mitochondria, and as a result, overall cellular fluorescence is diminished significantly.

Fluorescent Mitochondrial Membrane Depolarization Assays are for research use only. Not for use in diagnostic procedures.

JC-1
Mitochondrial depolarization
488 nm / 590 nm & 527 nm
Flow Cytometry, Fluorescence Microscope, Fluorescence Plate Reader
Cell culture
-20°C
Domestic: Overnight Delivery; International: Priority Shipping
United States
  1. Prepare samples.
  2. Create controls with CCCP.
  3. Dilute 10X Assay Buffer 1:10 with diH2O.
  4. Reconstitute MitoPT® JC-1 with DMSO.
  5. Dilute MitoPT® JC-1 with 1X Assay Buffer.
  6. Add MitoPT® JC-1 to each sample.
  7. Incubate 15-30 minutes.
  8. Wash cells: add 1X Assay Buffer and spin cells (twice).
  9. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. MitoPT® JC-1 excites at 488 nm. Aggregated MitoPT® JC-1 (orange) emits at 590 nm, monomeric MitoPT® JC-1 (green) emits at 527 nm.
Kit 911: 400 Tests
  • MitoPT® JC-1 Reagent, 400 Tests, #6260
  • 10X Assay Buffer (2 x 125 mL), #6259
  • CCCP, 50 mM, 600 µL, #6258
  • Kit Manual
    • Kit 924: 100 Tests
  • MitoPT® JC-1 Reagent, 100 Tests, #6261
  • 10X Assay Buffer, 60 mL, #685
  • CCCP, 50 mM, 125 µL, #6257
  • Kit Manual

    • Product Specific References

    PMID Publication
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    34193088Wu, W., et al. 2021. BCL-xL is Correlated With Disease Severity in Neonatal Infants With Sepsis. Research Square, 295.

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