Evaluation of Common Crosslinking Agents, Glutaraldehyde and EDC, for the Magnetic Isolation of Phagosomes from Cultured Retinal Pigment Epithelial Cells

Daily exposure of photoreceptors to light leads to damage and subsequent shedding of the photoreceptor outer segments (OS). The retinal pigment epithelium (RPE) is responsible for internalization, processing, and recycling of the shed photoreceptor OS. This phenomena, termed RPE phagocytosis, proceeds through a relatively unknown mechanism. To study RPE phagocytosis, previous studies labeled OS with magnetic nanoparticles through a glutaraldehyde cross‐linker to allow for magnetic isolation of phagosomes from the RPE. While isolation of phagosomes from the glutaraldehyde cross‐linking method was successful, the addition of this linker may impact the phagocytic ingestion pathway. Comparatively, 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), a zero‐length cross‐linker, may be used to create a direct cross‐link between the carboxylic acids of the OS protein and the amines of prepared magnetite particles. As such, the purpose of this study was to compare the outcomes of linking OS to nanomagnetites via glutaraldehyde and EDC. To prepare the nanomagnetites, Fe 3 O 4 particles coated with primary amine groups were centrifuged to separate nanoparticles from larger particles. The nanomagnetites were then linked to purified bovine OS with either glutaraldehyde or EDC for 3 hours or 15 minutes, respectively. Linked OS were then fed to cultured RPE cells for 4 hours. Cells were washed and then gently lysed using dounce homogenization to obtain intact phagosomes. Lysed cells were subjected to a magnetic field to separate phagosomes containing magnetite‐labeled OS from the remainder of the lysate. Immunoblot analysis of the purified phagosomes confirmed the presence of rhodopsin, a major component of OS, in both methods. In addition, isolated phagosomes from both methods were shown to contain proteins typically known to be associated with phagosomes including Rab 5, EEA1, and Annexin V. These results suggest that linkage of nanomagnetites to OS with either glutaraldehyde or EDC can serve as efficient methods to accomplish purification of phagosomes from the RPE. Due to the readily tunable linkage conditions, ease of use, and the formation of a direct peptide bond between the magnetite and OS, linkage of nanomagnetites to OS with EDC can serve as a more effective method for purification of phagosomes from RPE cells. Analysis of RPE phagosomes isolated by magnetic selection will contribute to the further elucidation of the RPE phagocytic process. Support or Funding Information Florida Southern College Department of Chemistry, Biochemistry, & Physics