HCS image showing nuclear segmentation and mitochondrial membrane potentialOptimized, automated assays for HCS

Membrane potential is a central feature of healthy mitochondria, and membrane depolarization is a good indicator of mitochondrial dysfunction, which is increasingly implicated in drug toxicity. This automated assay uses Invitrogen MitoTracker Orange as an indicator of mitochondrial function because its accumulation in the mitochondria of live cells is proportional to the mitochondrial membrane potential. Invitrogen Hoechst 33342 is used as a segmentation tool to identify cells; a viability stain, such as the Invitrogen Image-iT DEAD Green Viability Stain, is easily incorporated to further multiplex the assay. These three dyes have sufficient retention of fluorescence signal intensity upon formaldehyde fixation and detergent permeabilization to be useful in fixed endpoint assays, as well as applications involving immunocytochemistry for specific protein detection.

Typical mitochondrial health assay

Fixed-cell immunofluorescent assay protocol.

Imaging mode

  • Widefield
  • 10x or 20x magnification

Automatically measured properties

  • Mitochondrial membrane potential
  • Cell viability
  • Nuclear shape and dimensions

Mitochondrial health assay images

HepG2 cells stained with Invitrogen Hoechst 33342 stain to provide nuclear segmentation for automated cell counting and measures of nuclear morphology.

Invitrogen Image-iT DEAD Green stain used to label dead cells and provide a viability determination based on cell membrane permeability.

Invitrogen MitoTracker Orange used to label mitochondria with polarized membranes. In this assay, signal intensity in the orange channel is proportional to membrane potential and mitochondrial health.


Sample experimental data

Dose-response plot for HepG2 cells treated with increasing concentrations of valinomycin. Cells were stained with Invitrogen MitoTracker Orange to measure mitochondrial membrane potential, Invitrogen Hoechst 33342 dye to count total cells based on nuclear intensity, and Invitrogen Image-iT DEAD Green to identify dead cells. Automated imaging and analysis was done using Thermo Scientific ArrayScan VTI and the data were used to calculate EC50 values for cell loss, mitochondrial membrane potential, and membrane permeability using GraphPad Prism software.