Isolation of Phagosomes Containing Bovine Photoreceptor Outer Segments from Retinal Pigment Epithelial Cells by Magnetic Selection

Abstract

Daily phagocytosis of the distal tips of photoreceptor cells by the retinal pigment epithelium (RPE) is known to be an essential mechanism in the maintenance of the retina. The outermost portions of the photoreceptor cells, termed outer segments, are ingested and subsequently processed by the RPE. Previous studies involving the isolation of phagosomes have utilized latex beads to induce phagocytosis in macrophages, which lacks specificity due to the large foreign body being ingested. Therefore, the purpose of this study was to develop a standard protocol for the isolation of outer segment enriched phagosomes from RPE cells. To generate phagosomes with the selected cargo, outer segments isolated from bovine retinas were covalently labelled with magnetite particles and then fed to RPE‐J cells. After feeding, the cells were washed and gently lysed with a Dounce homogenizer. The phagosomes containing the magnetite bound outer segments were collected via magnetic rack isolation and stored in a homogenization solution. Immunoblots using antibodies against Mertk, N and C‐terminal rhodopsin, and cathepsin D confirmed the presence of outer segment‐containing phagosomes of varying maturity in the isolated fraction. The protocol we have developed has successfully isolated outer segment enriched phagosomes from RPE‐J cells by magnetic selection. This study differentiates itself from other studies that employ latex beads to isolate phagosomes through density gradient centrifugation. The isolation of these phagosomes will potentially play an important role in investigating protein interactions that impact RPE membrane trafficking and maturation.Support or Funding InformationFlorida Southern College Faculty‐Student Collaborative Research GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.