Time-lapse,single cell based confocal imaging analysis of caspase activation and phosphatidylserine flipping during cellular apoptosis

Abstract

Apoptosis is an important phenomenon for investigating the efficacy of anti-cancer drug candidates. The conventional assays for cellular apoptosis, such as enzyme-linked immunosorbent assay, absorbance monitoring for the activity of caspase, and flow cytometric assay, have focused only on biochemical events. We investigated the staurosporine (STS)-induced apoptosis of the murine macrophage RAW-264.7 cell using a cell based bioimaging technique. Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of green fluorescent protein from the cytosol to the nuclei. Five hours after 1 μM STS treatment, caspase-3 was observed to be activated and membrane blebbing was observed simultaneously. Also, the loss of phosphatidylserine (PS) asymmetry in the phospholipid bilayer of plasma membrane during early apoptosis was monitored by imaging annexin-V labeled with fluorescein isocyanate binding to the externalized PS at various concentrations of STS. Moreover, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. The single cell based bioimaging data agreed well with those of the biochemical assays for caspase activation and morphological observation for membrane integrity.