| Fluorescence-based assays designed to evaluate the functional status of mitochondria are emerging as
useful tools to elucidate the role of mitochondrial activity in the apoptosis cascade and other cellular
processes. Here we report the development of a live-cell multiparameter assay to measure mitochondrial
membrane potential and apoptosis based upon the Guava EasyCyte™ System, a blue laser 96-well
microcapillary flow cytometry instrument. In this assay, JC-1, a ratiometric dye that fluoresces either green
or orange depending upon membrane potential, is used to evaluate mitochondrial membrane potential
changes. A second dye, 7-Aminoactinomycin (7-AAD), a cell-impermeant DNA intercalator, is used to
simultaneously monitor cell membrane permeability changes commonly observed later in apoptosis,
as well as in necrotic cell death. We report this assay works for both adherent cells and suspension cells.
Using this assay we were able to calculate the EC50 for camptothecin-induced apoptosis in Jurkat cells upon
4 hour treatment at 0.5–1 mM, which is in good agreement to the EC50 measured by Annexin V staining.
Furthermore, using both JC-1 and 7-AAD in a single assay not only allows us to monitor cellular events
leading to both mitochondrial membrane potential change and apoptosis, but also enables us to monitor
certain cell culture conditions leading to mitochondrial membrane potential change without eliciting
apoptosis or vice versa. We conclude that in addition to monitoring apoptotic events within cells, this assay
provides users a method to measure the effects of subtle changes of cell culture conditions on cell health
and to separate cellular events leading to mitochondrial membrane potential change and apoptosis. |
