Multi-Parametric Analysis of Cell Death Pathways Using Live-Cell Microscopy

Programmed cell death is a complex process with new forms being discovered with regularity. Each pathway has a distinct and overlapping biochemical and physiological change occurring as the cell prepares for death. Detection of these changes can be facilitated by the availability of various fluorescent probes and advances in microscope systems. By analyzing these probes over time using fluorescence microscopy, the changes that occur in each cell en route to death can be analyzed on a cell-by-cell basis. While the timing of events varies considerably from cell to cell, it has been found that the sequence of events is highly conserved. Transient events, which would be difficult to detect using population averaging techniques, are readily detected when cells are analyzed individually in time lapse. The protocols in this unit describe using probes for real-time imaging of one of the apoptotic cell death pathways using various inducers, as well as the associated hardware necessary for imaging so that the imaging itself is not affecting cell viability. Curr. Protoc. Toxicol. 58:4.40.1-4.40.31. © 2013 by John Wiley & Sons, Inc. Keywords: apoptosis; fluorescence microscopy; real-time imaging; image analysis; FRET