Methods for studying apoptosis and phagocytosis of apoptotic cells in Drosophila tissues and cell lines

This chapter discusses the several techniques have been developed to label apoptotic cells in Drosophila tissues. Both acridine orange (AO) and the transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) technique can be used to assess apoptosis in the developing embryo and in the larval imaginal discs and nervous system. The molecular basis of AO staining is controversial but seems to involve specific retention of the dye in apoptotic cells. The TUNEL technique takes advantage of the DNA cleavage found in apoptotic cells. The free DNA ends targeted by terminal deoxytransferase (TdT), incorporates digoxygenin-labeled nucleotides. This chapter provides protocols for AO and TUNEL labeling, as well as for ultrastructural studies. The chapter discusses the other assays used for labeling apoptotic cells in Drosophila. Activation of caspases is a stereotypic feature of apoptotic cells and can be detected in situ with affinity-labeling techniques. The binding of Annexin V to exposed phosphatidylserine is another common technique used to label apoptotic cells. This chapter provides a protocol to assess embryonic phagocytosis of apoptotic cells. Phagocytosis also occurs later in development, a time when hemocytes are abundant and more accessible by bleeding.