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
In the Mitochondrial Potential Assay, cells are stained with Solution 7 and Solution 8, loaded into a cell counting slide, and counted with the NucleoCounter® NC-3000™.

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.

Data image from the Mitochondrial Potential Assay where the analyzed cells are shown in the NucleoView™ software.

Results presented in plot manager

Image of processed data from the Mitochondrial Potential Assay in the NucleoView™ software, in the form of histograms and scatter plots.

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.