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Vanessa Marie Morales, Sumana R Chintalapudi, Levon Djenderedjian, Jena J Steinle, Monica M Jablonski; Generation of a Novel Flow Cytometry-Based Protocol for the Enrichment and Isolation of Primary Murine Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3604.
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© ARVO (1962-2015); The Authors (2016-present)
Loss of functional retinal ganglion cells (RGC) is a key element of retinal degenerative diseases, yet the mechanisms underlying RGC degeneration are poorly understood. This is partly due to the lack of sufficient cellular material for in-depth analysis of ganglion cell pathways. Here, we developed a standardized protocol for highly enriched RGC isolation for future generation of an authenticated cell line.
Our protocol utilizes positive and negative selection of different surface markers, including anti-Thy and anti-CD48 antibodies. Additional cell surface markers, characteristic of other retinal cells, were included to eliminate contaminant cell types. Pre- and post-sorting analyses were performed to determine the purity of the enriched population. Validation and authentication analyses were performed by genomic profiling and intracellular detection of RGC-associated proteins, including RBPMS and g-synuclein.
Each individual mouse retina yielded 5x106±1.2 cells on average. Consistently, these were 95.0±2.4% alive by exclusion of live dead aqua (LDA) viability dye. We found in mice, anti-Thy and anti-CD48 antibodies are not sufficient to identify RGCs, as only 50% of cells showed g-synuclein positivity. Our enrichment procedure generated a homogeneous population with a sort efficiency of >97%. We used a very stringent validation method including gene expression analyses to determine population homogeneity and more importantly, contamination levels by other retinal cells.
A number of laboratories have emphasized the need for the development of a RGC line to study the molecular pathways involved in cell death. We have developed a novel protocol for the isolation of highly enriched RGCs using flow cytometry. Our method provides the starting material with which to generate an immortalized cell line by transforming this homogeneous population of RGCs.
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