Hazim Roni A, Williams David S
Department of Ophthalmology, Stein Eye Institute, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA, USA.
Neuroscience Interdepartmental Ph.D. Program, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, CA, USA.
Methods Mol Biol. 2018;1753:63-71. doi: 10.1007/978-1-4939-7720-8_4.
The phagocytosis of photoreceptor outer segments (POSs) by the retinal pigment epithelium (RPE) is essential for retinal homeostasis. Defects in this process can be caused by mutations in the photoreceptor cells, the RPE cells, or both cell types. This function can be experimentally investigated by performing an in vitro phagocytosis assay, in which cultured RPE cells are challenged with isolated POSs, and subsequently tested for their ability to degrade the POSs. A significant advantage of this approach is that mutant phenotypes can be attributed either to the photoreceptor or the RPE cells, by experimenting with different permutations of mutant and control photoreceptor and RPE cells. In this chapter, we detail the method for a double-immunofluorescence assay for analysis of the binding, ingestion, and subsequent degradation of isolated mouse POSs by cultured mouse primary RPE cells.
视网膜色素上皮（RPE）对光感受器外段（POSs）的吞噬作用对于视网膜内环境稳定至关重要。此过程中的缺陷可能由光感受器细胞、RPE细胞或两种细胞类型的突变引起。该功能可通过体外吞噬试验进行实验研究，在该试验中，用分离的POSs刺激培养的RPE细胞，随后测试其降解POSs的能力。这种方法的一个显著优点是，通过对突变型和对照光感受器及RPE细胞进行不同组合的实验，突变表型可归因于光感受器或RPE细胞。在本章中，我们详细介绍了一种双免疫荧光分析方法，用于分析培养的小鼠原代RPE细胞对分离的小鼠POSs的结合、摄取及随后的降解情况。
