Measuring apoptosis at the single cell level

Abstract

The use of live cell microscopy has made a number of contributions to the study of apoptosis. Many of the tools and techniques are available that allow us to image the key events that occur during cell death including mitochondrial outer membrane permeabilization, mitochondrial transmembrane potential changes, translocation of Bcl-2 family members, caspase activation, phosphatidylserine flip and plasma membrane rupture. We discuss these techniques here and highlight the advantages and drawbacks of using such approaches to study apoptosis.

Figure 1

Time-lapse analysis of the morphological changes that occur during apoptosis. Hela cyt c-GFP cells were stained with TMRE (50nM, red), Annexin V-APC (1% (v/v), blue) and PI (0.4µg/ml, pink) and treated with actinomycin D (1µM). The time after exposure to actinomycin D is at the top left of each panel. Scale bars (10µm) are at the bottom right of each panel.

Figure 2

Time-lapse analysis of the relative brightness of TMRE compared to the punctate diffuse index of cytochrome c-GFP. Hela cyt c-GFP cells were exposed to Act D (0.5µM) and the caspase inhibitor qVD-OPH (20µM) for 13 hours. Each point on the graph represents the average punctate/diffuse index or average relative TMRE fluorescence of 32 cells at 3 min intervals. Error bars represent the SEM.