Detection of Cell Death and Phagocytosis in the Drosophila Ovary
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Billions of cells die and are cleared throughout the development and homeostasis of an organism. Either improper death or clearance can lead to serious illnesses. In the adult Drosophila ovary, germline cells can die by programmed cell death (PCD) at three distinct stages; here we focus on cell death that occurs in mid- and late oogenesis. In mid-oogenesis, the germline of egg chambers can undergo apoptosis in response to nutrient deprivation. In late oogenesis, the nurse cells are eliminated through a developmentally regulated, non-apoptotic cell death. In this chapter, we describe several methods to detect cell death and phagocytosis in the Drosophila ovary. DAPI stains the chromatin of all cells and can be used to detect morphological changes in cells that die by different mechanisms. TUNEL labels fragmented DNA, which can occur in both apoptotic and non-apoptotic death. LysoTracker, an acidophilic dye, marks acidic vesicles and some dying cells; therefore, it can be used to study both death and phagocytosis. We also describe several antibodies that can be used to investigate cell death and/or phagocytosis: active caspase Dcp-1, membrane markers, and lamins. Many of these antibodies can be used in combination with GFP fusion transgenes for further analysis; we show Rab5-GFP and Rab7-GFP, which can be used to study phagocytosis in further detail.
KeywordsDrosophila Ovary Cell death Apoptosis Phagocytosis DAPI TUNEL LysoTracker
We obtained antibodies from the Developmental Studies Hybridoma Bank developed under the auspices of the NIHCD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242. We would also like to thank the Bloomington Stock Center, FlyTrap, and Trudi Schϋpbach for flies, and NIH RO1 grants GM060574 and GM094452 for funding. Lastly, we would like to thank members of the McCall lab for edits and discussions.
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