Optimized, 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.
Mitochondrial health assay images
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 HCS Platform and the data were used to calculate EC50 values for cell loss, mitochondrial membrane potential, and membrane permeability using GraphPad Prism software.
For Research Use Only. Not for use in diagnostic procedures.