Protocol

Retinal Degeneration

Volume 935 of the series Methods in Molecular Biology pp 271-283

Date:

Functional Analysis of Retinal Microglia and Their Effects on Progenitors

  • Debra A. CarterAffiliated withBristol Eye Hospital, School of Clinical Sciences, University of Bristol
  • , Balini BalasubramaniamAffiliated withBristol Eye Hospital, School of Clinical Sciences, University of Bristol
  • , Andrew D. DickAffiliated withBristol Eye Hospital, School of Clinical Sciences, University of Bristol Email author 

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Abstract

The identification of stem/progenitor cells within the retinal neural environment has opened up the possibility of therapy via cellular replacement and/or reprogramming of resident cell populations (1–4). Within the neuro-retinal niche, following injury or in disease states (including inflammation and degeneration), cellular responses affect tissue homeostasis, reduce cell density, disrupt tissue architecture, and produce scar formation. Microglia (resident retinal immune cell tissue macrophage) are key to the maintenance of retinal homeostasis and are implicated in responses that may influence the control and behavior of retinal progenitors (5, 6). Factors to consider in the generation of a transplantable cell resource with good migratory and integrative capacity include their yield, purity, and functional viability. Utilizing human postmortem retina, we have created a research platform to isolate, culture, and characterize adult retinal microglia as well as analyze their effect on retinal progenitors. Here, we describe techniques using magnetic labeled bead cell separation to isolate pure populations of retinal CD133+ precursor cells and CD11b+ microglia from primary adult retinal cell suspensions (RCSs), enabling flow cytometric cell phenotypic and qPCR genotypic analysis, as well as functional analysis by real-time ratiometric calcium imaging.

Key words

Primary retinal cells CD133 CD11b Magnetic labeled cell separation Flow cytometry RNA isolation qPCR Calcium imaging