Cell Culture Analysis of the Phagocytosis of Photoreceptor Outer Segments by Primary Mouse RPE Cells

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1753)

Abstract

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.

Key words

Phagocytosis Retinal pigment epithelium Photoreceptor outer segments Phagosome degradation Retinal homeostasis 

Notes

Acknowledgments

This study was supported by NIH grants R01EY13408 and P30EY00331 (DSW), and F31EY026805 (RAH).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Ophthalmology, Stein Eye InstituteDavid Geffen School of Medicine at University of California Los AngelesLos AngelesUSA
  2. 2.Neuroscience Interdepartmental Ph.D. ProgramDavid Geffen School of Medicine at University of California Los AngelesLos AngelesUSA
  3. 3.Department of NeurobiologyDavid Geffen School of Medicine at University of California Los AngelesLos AngelesUSA
  4. 4.Brain Research InstituteDavid Geffen School of Medicine at University of California Los AngelesLos AngelesUSA

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