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

 Samin Hong  ;  Yoko Iizuka  ;  Chan Yun Kim  ;  Gong Je Seong 
 Molecular Vision, Vol.18 : 2922-2930, 2012 
Journal Title
 Molecular Vision 
Issue Date
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.
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1. Journal Papers (연구논문) > 1. College of Medicine (의과대학) > Dept. of Ophthalmology (안과학교실)
1. Journal Papers (연구논문) > 1. College of Medicine (의과대학) > Yonsei Biomedical Research Center (연세의생명연구원)
Yonsei Authors
김찬윤(Kim, Chan Yun) ; 성공제(Seong, Gong Je) ; 이주카요코(Iizuka, Yoko) ; 홍사민(Hong, Sa Min)
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