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).
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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
Price:
$509.90
Ships: 1-2 business days
  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

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