Isolation of primary mouse retinal ganglion cells using immunopanning-magnetic separation

[摘要] Purpose: To establish an effective system for isolating primary retinal ganglion cells (RGCs) from newborn mice.Methods: The retinas were separated from enucleated eyeballs of Crl:CD-1 mice on postnatal day 1 to 4. RGCs were purified using three different methods, including two-step immunopanning (TSI), direct magnetic separation (DMS), and immunopanning-magnetic separation (IMS). Harvested cells were maintained for 24 h in a defined medium and then examined with immunocytochemistry, western immunoblotting, and real-time reverse transcription polymerase chain reaction (RT-PCR) for glial cell–specific glial fibrillary acidic protein (GFAP) and amacrine cell-specific syntaxin 1.Results: As determined with immunofluorescence staining, RGCs purified by TSI were sparsely mixed with GFAP-positive astrocytes, and RGCs isolated by DMS were frequently mixed with syntaxin 1-positive amacrine cells. However, RGCs collected by IMS were seldom contaminated by GFAP-positive or syntaxin 1-positive cells. On western immunoblots, TSI cells showed significant GFAP expression, and DMS cells showed apparent syntaxin 1 expression, but IMS cells did not. Results of the real-time RT–PCR showed a similar tendency to those of the immunocytochemistry and western immunoblots.Conclusion: Primary mouse RGCs were highly purified by the IMS method, combining the benefits of the TSI and DMS methods. This isolation method may provide a good experimental system for studying glaucoma in vitro.

[学科分类] 生物化学/生物物理