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Culture and Characterization of Microglia from the Adult Murine Retina

Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells. Method. Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seed...

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Autores principales: Devarajan, Gayathri, Chen, Mei, Muckersie, Elizabeth, Xu, Heping
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060747/
https://www.ncbi.nlm.nih.gov/pubmed/24987746
http://dx.doi.org/10.1155/2014/894368
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author Devarajan, Gayathri
Chen, Mei
Muckersie, Elizabeth
Xu, Heping
author_facet Devarajan, Gayathri
Chen, Mei
Muckersie, Elizabeth
Xu, Heping
author_sort Devarajan, Gayathri
collection PubMed
description Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells. Method. 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. Conclusion. We have developed a simple method for isolating and culturing retinal microglia from adult mice.
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spelling pubmed-40607472014-07-01 Culture and Characterization of Microglia from the Adult Murine Retina Devarajan, Gayathri Chen, Mei Muckersie, Elizabeth Xu, Heping ScientificWorldJournal Research Article Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells. Method. 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. Conclusion. We have developed a simple method for isolating and culturing retinal microglia from adult mice. Hindawi Publishing Corporation 2014 2014-05-29 /pmc/articles/PMC4060747/ /pubmed/24987746 http://dx.doi.org/10.1155/2014/894368 Text en Copyright © 2014 Gayathri Devarajan et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Devarajan, Gayathri
Chen, Mei
Muckersie, Elizabeth
Xu, Heping
Culture and Characterization of Microglia from the Adult Murine Retina
title Culture and Characterization of Microglia from the Adult Murine Retina
title_full Culture and Characterization of Microglia from the Adult Murine Retina
title_fullStr Culture and Characterization of Microglia from the Adult Murine Retina
title_full_unstemmed Culture and Characterization of Microglia from the Adult Murine Retina
title_short Culture and Characterization of Microglia from the Adult Murine Retina
title_sort culture and characterization of microglia from the adult murine retina
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060747/
https://www.ncbi.nlm.nih.gov/pubmed/24987746
http://dx.doi.org/10.1155/2014/894368
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