title: Isolation of primary mouse retinal ganglion cells using immunopanning-magnetic separation.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. samini@yuhs.ac
authors.forename: Samin
authors.initials: S
authors.lastname: Hong
authors.forename: Yoko
authors.initials: Y
authors.lastname: Iizuka
authors.forename: Chan Yun
authors.initials: CY
authors.lastname: Kim
authors.forename: Gong Je
authors.initials: GJ
authors.lastname: Seong
mesh_terms: D025042:Amacrine Cells / Q000166:cytology / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D005260:Female; D015870:Gene Expression; D005904:Glial Fibrillary Acidic Protein; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation / Q000379:methods* / Q000592:standards; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D060888:Real-Time Polymerase Chain Reaction; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D015415:Biomarkers; D005904:Glial Fibrillary Acidic Protein; D009419:Nerve Tissue Proteins; D050827:Syntaxin 1; C528900:glial fibrillary astrocytic protein, mouse