Methods and protocols for studying cell death in Drosophila.
Drosophila melanogaster is a highly amenable model system for examining programmed cell death during animal development, offering sophisticated genetic techniques and in vivo cell biological analyses. The reproducible pattern of apoptosis, as well as the apoptotic response to genotoxic stress, has been well characterized during Drosophila development. The main cellular components required for cell death are highly conserved throughout evolution. Central to the regulation of apoptosis is the caspase family of cysteine proteases, and studies in Drosophila have revealed insights into their regulation and function. This chapter describes protocols for detecting apoptotic cells during Drosophila development, as well as the use of Drosophila cell lines. Commonly used methods for detecting apoptosis are described, including TUNEL, acridine orange, and immunostaining with specific components of the apoptotic pathway such as active caspases. A crucial step in the induction of apoptosis is caspase activation and cleavage, which can be measured by use of fluorogenic peptide substrates or detection of cleaved protein products by immunoblotting, respectively. In addition, one of the advantages of the use of Drosophila as model is the ability to examine genetic interactions with various components of the cell death pathway.
Denton
Donna
D
Hanson Institute, Institute of Medical and Veterinary Science, Adelaide, Australia.
Mills
Kathryn
K
Kumar
Sharad
S
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3 / Q000032:analysis; D002460:Cell Line; D045304:Cytochromes c / Q000032:analysis; D004331:Drosophila melanogaster / Q000737:chemistry / Q000166:cytology* / Q000235:genetics / Q000254:growth & development; D005123:Eye / Q000378:metabolism; D020287:In Situ Nick-End Labeling / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D019076:Transgenes
D045304:Cytochromes c; D053148:Caspase 3