Meehan Tracy L, Yalonetskaya Alla, Joudi Tony F, McCall Kimberly
Department of Biology, Boston University, Boston, MA, 02215, USA.
Methods Mol Biol. 2015;1328:191-206. doi: 10.1007/978-1-4939-2851-4_14.
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.
在生物体的发育和内稳态过程中，数十亿细胞死亡并被清除。细胞死亡或清除不当都可能导致严重疾病。在成年果蝇卵巢中，生殖细胞可在三个不同阶段通过程序性细胞死亡（PCD）死亡；在此我们聚焦于卵子发生中期和后期发生的细胞死亡。在卵子发生中期，卵室的生殖细胞可因营养剥夺而发生凋亡。在卵子发生后期，滋养细胞通过一种发育调控的非凋亡性细胞死亡被清除。在本章中，我们描述了几种检测果蝇卵巢中细胞死亡和吞噬作用的方法。DAPI可对所有细胞的染色质进行染色，可用于检测因不同机制死亡的细胞的形态变化。TUNEL标记断裂的DNA，其可发生在凋亡性和非凋亡性死亡中。溶酶体示踪剂（LysoTracker）是一种嗜酸性染料，可标记酸性囊泡和一些正在死亡的细胞；因此，它可用于研究细胞死亡和吞噬作用。我们还描述了几种可用于研究细胞死亡和/或吞噬作用的抗体：活性半胱天冬酶Dcp-1抗体、膜标记物抗体和核纤层蛋白抗体。这些抗体中的许多可与绿色荧光蛋白（GFP）融合转基因结合使用以进行进一步分析；我们展示了Rab5-GFP和Rab7-GFP，它们可用于更详细地研究吞噬作用。
