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Abstract #51958 Published in IGR 14-4
Isolation of primary mouse retinal ganglion cells using immunopanning-magnetic separation
Hong S; Iizuka Y; Kim CY; Seong GJMolecular Vision 2012; 18: 2922-2930
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.
Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. samini@yuhs.ac
Classification:
3.6 Cellular biology (Part of: 3 Laboratory methods)
5.1 Rodent (Part of: 5 Experimental glaucoma; animal models)
