In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina

Abstract

microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.

Phenotypes of primary retinal macroglia: Implications for purification and culture conditions

“…We have previously published methods for isolating Müller glia and retinal astrocytes as separate cell populations, using immunomagnetic separation (Sappington et al, 2006a(Sappington et al, , 2006bLee et al, 2015). This antigen-based method of cell separation has been utilized routinely by us and others for studies examining everything from cell signaling and behavior to high-throughput proteomic and genomic screens (Scheef et al, 2005;Ho et al, 2014;Hosoki et al, 2015;Reyes-Aguirre et al, 2018). The goal of this study was to characterize astrocyte and Müller cell phenotypes induced by previously published culture conditions, particularly those utilized for high-throughput screening.…”