Real-Time visualization of caspase-3 activation by fluorescence resonance energy transfer (FRET)

Abstract

As apoptosis occurs via a complex signaling cascade that is tightly regulated at multiple cell points, different methods exist to evaluate the activity of the proteins involved in the intracellular apoptotic pathways and the phenotype of apoptotic neurons. Detention of the activity of the enzyme caspase-3, the key executioner caspase in programmed cell death, by laser scanning confocal fl uorescence microscopy and the fl uorescence resonance energy transfer technology is an alternative approach to classical standard techniques, such as Western blotting, activity assays, or histological techniques, and allows working with both fi xed and living cells. This technique combined with the organotypic culture approach ex vivo represents a valid tool for the study of the mechanisms of neuronal survival/death and neuroprotection.