Limits...
Culture and characterization of microglia from the adult murine retina.

Devarajan G, Chen M, Muckersie E, Xu H - ScientificWorldJournal (2014)

Bottom Line: 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.We have developed a simple method for isolating and culturing retinal microglia from adult mice.

View Article: PubMed Central - PubMed

Affiliation: Infection and Immunity, School of Medicine, University of Aberdeen, Foresterhill AB25 2ZD, UK.

ABSTRACT

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.

Show MeSH

Related in: MedlinePlus

Effects of different culture media on the yield and the morphology of the cultured microglial cells. (a) Mixed retinal culture grown in DMEM showing low cell numbers. ((b) and (d)) Cells grown in DMEM/F12 + Glutamax showing a good yield and no change in the morphology was observed. ((c) and (e)) Some of the cells grown in RPMI/F12 + Glutamax started to change into more round-shaped cells. Microglial cells were observed after 15 days in culture using a phase-contrast Olympus microscope and Prog Res C14 imaging software. Magnification ×20; (d) and (e) are enlarged. Data are representative of 3 experimental repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4060747&req=5

fig1: Effects of different culture media on the yield and the morphology of the cultured microglial cells. (a) Mixed retinal culture grown in DMEM showing low cell numbers. ((b) and (d)) Cells grown in DMEM/F12 + Glutamax showing a good yield and no change in the morphology was observed. ((c) and (e)) Some of the cells grown in RPMI/F12 + Glutamax started to change into more round-shaped cells. Microglial cells were observed after 15 days in culture using a phase-contrast Olympus microscope and Prog Res C14 imaging software. Magnification ×20; (d) and (e) are enlarged. Data are representative of 3 experimental repeats.

Mentions: Culture Medium. To identify the media that are best suited for microglial cells, we tested DMEM, RPMI/F12 + Glutamax or DMEM/F12 + Glutamax. The mixed retinal cells were seeded in a 24-well plate at the density of 0.75 million cells per cm2 and supplemented with FCS and L929 supernatant. Cells cultured in DMEM failed to proliferate (Figure 1(a)), whereas cells grown in DMEM/F12 + Glutamax and RPMI/F12 + Glutamax proliferated (Figures 1(b) and 1(c), resp.). Microglial cells cultured in DMEM/F12 + Glutamax resembled a more ramified shape, whereas cells grown in RPMI/F12 + Glutamax have macrophage-like amoeboid morphology (Figures 1(d) and 1(e), resp.). Therefore, DMEM/F12 + Glutamax provides an optimum condition for the growth and maintenance of retinal microglia.


Culture and characterization of microglia from the adult murine retina.

Devarajan G, Chen M, Muckersie E, Xu H - ScientificWorldJournal (2014)

Effects of different culture media on the yield and the morphology of the cultured microglial cells. (a) Mixed retinal culture grown in DMEM showing low cell numbers. ((b) and (d)) Cells grown in DMEM/F12 + Glutamax showing a good yield and no change in the morphology was observed. ((c) and (e)) Some of the cells grown in RPMI/F12 + Glutamax started to change into more round-shaped cells. Microglial cells were observed after 15 days in culture using a phase-contrast Olympus microscope and Prog Res C14 imaging software. Magnification ×20; (d) and (e) are enlarged. Data are representative of 3 experimental repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4060747&req=5

fig1: Effects of different culture media on the yield and the morphology of the cultured microglial cells. (a) Mixed retinal culture grown in DMEM showing low cell numbers. ((b) and (d)) Cells grown in DMEM/F12 + Glutamax showing a good yield and no change in the morphology was observed. ((c) and (e)) Some of the cells grown in RPMI/F12 + Glutamax started to change into more round-shaped cells. Microglial cells were observed after 15 days in culture using a phase-contrast Olympus microscope and Prog Res C14 imaging software. Magnification ×20; (d) and (e) are enlarged. Data are representative of 3 experimental repeats.
Mentions: Culture Medium. To identify the media that are best suited for microglial cells, we tested DMEM, RPMI/F12 + Glutamax or DMEM/F12 + Glutamax. The mixed retinal cells were seeded in a 24-well plate at the density of 0.75 million cells per cm2 and supplemented with FCS and L929 supernatant. Cells cultured in DMEM failed to proliferate (Figure 1(a)), whereas cells grown in DMEM/F12 + Glutamax and RPMI/F12 + Glutamax proliferated (Figures 1(b) and 1(c), resp.). Microglial cells cultured in DMEM/F12 + Glutamax resembled a more ramified shape, whereas cells grown in RPMI/F12 + Glutamax have macrophage-like amoeboid morphology (Figures 1(d) and 1(e), resp.). Therefore, DMEM/F12 + Glutamax provides an optimum condition for the growth and maintenance of retinal microglia.

Bottom Line: 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.We have developed a simple method for isolating and culturing retinal microglia from adult mice.

View Article: PubMed Central - PubMed

Affiliation: Infection and Immunity, School of Medicine, University of Aberdeen, Foresterhill AB25 2ZD, UK.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus