Cellular organelle mitochondria

Mitochondrial Potential Assay

Assessing Apoptosis Using the Lipophilic Dye JC-1

  • Easily distinguish polarized (healthy) cells, depolarized (apoptotic) cells and necrotic/late-stage apoptotic cells
  • Fast, automated single cell analysis
  • Acquisition and analysis in one simple step
  • Standardized results – even with different users
  • No calibration required
  • Clear data presentation with Plot Manager feature
  • Automated PDF reports
  • Export data in FCS/ACS formats

Overview of the Mitochondrial Potential Assay

The lipophilic cationic dye JC-1 can be used to evaluate the mitochondrial potential. In healthy cells, the negative charge established by the intact mitochondrial membrane potential facilitates the accumulation of JC-1 in the mitochondrial matrix.

Loss of the mitochondrial membrane potential is known to precede apoptosis and chemical hypoxia-induced necrosis. JC-1 displays potential-dependent accumulation in the mitochondria and has dual-fluorescent properties: Red fluorescence indicates the dye has formed aggregates, while green fluorescence indicates the dye is monomeric. This is a simple, fluorescent-based method for distinguishing healthy cells (staining red) from apoptotic cells (staining green) in a sample.

Assay Principle

Using fluorescence microscopy and image analysis, the NucleoCounter® NC-3000™ advanced image cytometer automatically detects cells with collapsed mitochondrial membrane potential. Cells are stained with JC-1 and DAPI (Solution 7 and Solution 8, respectively).

Cellular JC-1 monomers and aggregates are detected as green and red fluorescence intensity, respectively. Mitochondrial depolarization is revealed as a decrease in the red/green fluorescence ratio. Necrotic and late apoptotic cells are detected as blue fluorescent (i.e. DAPI positive) cells.

Annexin V Assay mitochondrial potential

Results Presented in Plot Manager

Graphs acquired using plot manager feature with NC-3000 and Mitochondrial Potential Assay

The diagram demonstrates the effect of adding camptothecin (CPT) on Jurkat cells. CPT is a commonly used topoisomerase poison, which induces apoptosis, thus disrupting the mitochondrial potential of cells. Jurkat cells are an immortalized line of human T lymphocyte cells, commonly used for studying the effectiveness of potential anti-cancer compounds.

Jurkat cells were grown in the absence (upper row) or in the presence (lower row) of camptothecin (CPT). Cells were stained with JC-1 and DAPI and analyzed using the Mitochondrial Potential Assay. Scatter plots and histograms were obtained from the NucleoView™ NC-3000™ software.
Polygons and markers in the displayed plots are used to demarcate the various cell populations. In this example, 9% of untreated cells are depolarized/apoptotic, whereas 61% of CPT-treated cells are depolarized/apoptotic. These findings suggest that CPT has a strong effect on mitochondrial potential.

Publications

MIM1, the Mcl-1 – specific BH3 mimetic induces apoptosis in human U87MG glioblastoma cells

  • Respondek M, Beberok A, Rok J, et al. ,

  • Toxicol In Vitro ,

  • 2018,

  • 53:126-135

  • Instrument: NucleoCounter® NC-3000™
  • Cell Type: Cancer Cells
  • Research area: Small Molecule Drugs
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