A new technique for real-time analysis of caspase-3 dependent neuronal cell death.

摘要

Several markers are available to identify cells undergoing programmed cell death, but so far they are only applicable on fixed material. Therefore, no information on the kinetics of apoptosis can be obtained, although apoptosis is a dynamic cell process. Here, we describe a new technique that allows the real-time observation of the onset of apoptosis in primary neurons. Neurons are transfected with a plasmid that codes for a fluorescent protein localized in the soma. Upon activation of caspase-3, which represents the point-of-no-return in the apoptosis process, the fusion protein is cleaved and as a consequence translocates into the nucleus. The onset of apoptosis is thus visualized by translocation of the fluorescent signal from the soma to the nucleus. The translocation process was found to be specific for the apoptosis process as it correlates with the activation of caspase-3 and TUNEL staining. This tool does not require complex detection systems and allows for the first time the analysis of the kinetics of apoptosis in a simple and efficient manner.