Use of Necrotic Markers in the Drosophila Ovary

Abstract

Necrosis is a form of cell death characterized by cytoplasmic and organelle swelling, compromised ­membrane integrity, intracellular acidification, and increased levels of reactive oxygen species (ROS) and cytosolic Ca²⁺. In the Drosophila ovary, two distinct forms of cell death occur naturally. In response to starvation, caspase-dependent cell death occurs during mid-oogenesis. Additionally, the nurse cells, which support the develo** oocyte, undergo developmental programmed cell death during late oogenesis after they dump their contents into the oocyte. Evidence suggests that necrosis may be playing an important role during developmental programmed cell death of the nurse cells during late oogenesis. Here, we describe several methods to detect events associated with necrosis in the Drosophila ovary. Propidium iodide is used to detect cells with compromised membrane integrity, and H₂DCFDA is used as an indicator of ROS levels in a cell. In addition, LysoTracker detects intracellular acidification and X-rhod-1 detects cytosolic Ca²⁺. We also describe transgenic methods to detect Ca²⁺ levels and expression patterns. These methods performed in the Drosophila ovary, as well as other tissues, may lead to a further understanding of the mechanisms of necrosis as a form of programmed cell death.