title: Culture and characterization of microglia from the adult murine retina.
abstract: OBJECTIVE
  To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells.
  
  
  METHODS
  Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seeding cell density, and purification of microglia from the mixed cultures were optimised. Cultured retinal microglial cells were phenotyped using the surface markers CD45, CD11b, and F4/80. Their ability to secrete proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation was examined using cytometric bead array (CBA) assay.
  
  
  RESULTS
  Higher yield was obtained when retinal single-cell suspension was cultured at the density of 0.75 × 10(6) cells per cm(2) in Dulbecco's modified Eagle medium (DMEM)/F12 + Glutamax supplement with 20% fetal calf serum (FCS) and 20% L929 supernatant. We identified day 10 to be the optimum day of microglial isolation. Over 98% of the cells isolated were positive for CD45, CD11b, and F4/80. After stimulating with LPS they were able to secrete proinflammatory cytokines such as IL-6 and TNF- α and express CD86, CD40, and MHC-II.
  
  
  CONCLUSIONS
  We have developed a simple method for isolating and culturing retinal microglia from adult mice.
authors.affiliation: Infection and Immunity, School of Medicine, University of Aberdeen, Foresterhill AB25 2ZD, UK.
authors.forename: Gayathri
authors.identifier: 0000-0001-5055-0519
authors.initials: G
authors.lastname: Devarajan
authors.affiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Institute of Clinical Science-A, Grosvenor Road, Belfast BT12 6BA, UK.
authors.forename: Mei
authors.initials: M
authors.lastname: Chen
authors.affiliation: Infection and Immunity, School of Medicine, University of Aberdeen, Foresterhill AB25 2ZD, UK.
authors.forename: Elizabeth
authors.identifier: 0000-0003-4674-6214
authors.initials: E
authors.lastname: Muckersie
authors.affiliation: Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Institute of Clinical Science-A, Grosvenor Road, Belfast BT12 6BA, UK.
authors.forename: Heping
authors.identifier: 0000-0003-4000-931X
authors.initials: H
authors.lastname: Xu
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D061251:Primary Cell Culture / Q000379:methods*; D012160:Retina / Q000166:cytology*
chemical_list: D003470:Culture Media; D016207:Cytokines