Timmons Allison K, Meehan Tracy L, Gartmond Tori D, McCall Kimberly
Department of Biology, Boston University, Boston, MA, USA.
Methods Mol Biol. 2013;1004:215-28. doi: 10.1007/978-1-62703-383-1_16.
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 Ca(2+). 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 developing 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 Ca(2+). We also describe transgenic methods to detect Ca(2+) 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.
坏死是一种细胞死亡形式，其特征为细胞质和细胞器肿胀、膜完整性受损、细胞内酸化以及活性氧（ROS）和胞质Ca(2+)水平升高。在果蝇卵巢中，自然发生两种不同形式的细胞死亡。在饥饿应激下，半胱天冬酶依赖性细胞死亡发生在卵子发生中期。此外，支持发育中卵母细胞的滋养细胞在卵子发生后期将其内容物排入卵母细胞后，会经历发育程序性细胞死亡。有证据表明，坏死可能在卵子发生后期滋养细胞的发育程序性细胞死亡过程中发挥重要作用。在此，我们描述了几种检测果蝇卵巢中与坏死相关事件的方法。碘化丙啶用于检测膜完整性受损的细胞，H2DCFDA用作细胞内ROS水平的指示剂。此外，溶酶体示踪剂检测细胞内酸化，X-rhod-1检测胞质Ca(2+)。我们还描述了检测Ca(2+)水平和表达模式的转基因方法。这些在果蝇卵巢以及其他组织中进行的方法可能会进一步加深我们对坏死作为一种程序性细胞死亡形式的机制的理解。
