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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.

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Immunofluorescent staining of retinal microglial cells. (a) Microglial cells were transferred into a 16-well chamber slide and stimulated with LPS in the absence of L929 supernatant for 24 h. After stimulation, cells were stained for activation markers, including MHC-II (FITC), CD86 (PE), and CD40 (APC). The slides were observed by confocal microscopy. (b) Microglial cells were transferred into a 16-well chamber slide, incubated with POS-FITC at the ratio of cells to POS-FITC 1 : 5 for 18 h, and stained for CD11b (APC). (c) Microglial cells (1 × 104/well) were transferred into a 96-well plate and stimulated with 1 μg/mL of LPS for 24 h. The supernatant was collected and measured for the presence of IL-1β, IL-12, IL-10, CCL2, GM-CSF, IL-6, TNF-α, and CCL5 using CBA. *P ≤ 0.05 compared with control nonstimulated cell supernatant. Mean ± SEM, n = 3.
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fig6: Immunofluorescent staining of retinal microglial cells. (a) Microglial cells were transferred into a 16-well chamber slide and stimulated with LPS in the absence of L929 supernatant for 24 h. After stimulation, cells were stained for activation markers, including MHC-II (FITC), CD86 (PE), and CD40 (APC). The slides were observed by confocal microscopy. (b) Microglial cells were transferred into a 16-well chamber slide, incubated with POS-FITC at the ratio of cells to POS-FITC 1 : 5 for 18 h, and stained for CD11b (APC). (c) Microglial cells (1 × 104/well) were transferred into a 96-well plate and stimulated with 1 μg/mL of LPS for 24 h. The supernatant was collected and measured for the presence of IL-1β, IL-12, IL-10, CCL2, GM-CSF, IL-6, TNF-α, and CCL5 using CBA. *P ≤ 0.05 compared with control nonstimulated cell supernatant. Mean ± SEM, n = 3.

Mentions: To characterise the cells, the retinal cultures were transferred into a chamber slide and stained for the surface markers including CD11b, F4/80, and CD45. None of the above markers are specific to the retinal microglial cells. However, microglia are generally characterised as CD11bhigh and CD45low. The majority (>98%) of the cultured cells were positive for CD11b indicating that the cells belong to the mononuclear phagocytic system. The cells expressed high levels of F4/80 (Figure 5(b)) and CD11b (Figure 5(c)) and low levels of CD45 (Figure 5(d)). The cultured retinal microglial cells were negative for MHC-II, CD86, and CD40 (Figures 5(e), 5(f), and 5(g), resp.) indicating that they were not activated. However, microglial cells were positive for MHC-II, CD86, and CD40 in response to LPS stimulation (Figure 6(a)).


Culture and characterization of microglia from the adult murine retina.

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

Immunofluorescent staining of retinal microglial cells. (a) Microglial cells were transferred into a 16-well chamber slide and stimulated with LPS in the absence of L929 supernatant for 24 h. After stimulation, cells were stained for activation markers, including MHC-II (FITC), CD86 (PE), and CD40 (APC). The slides were observed by confocal microscopy. (b) Microglial cells were transferred into a 16-well chamber slide, incubated with POS-FITC at the ratio of cells to POS-FITC 1 : 5 for 18 h, and stained for CD11b (APC). (c) Microglial cells (1 × 104/well) were transferred into a 96-well plate and stimulated with 1 μg/mL of LPS for 24 h. The supernatant was collected and measured for the presence of IL-1β, IL-12, IL-10, CCL2, GM-CSF, IL-6, TNF-α, and CCL5 using CBA. *P ≤ 0.05 compared with control nonstimulated cell supernatant. Mean ± SEM, n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Immunofluorescent staining of retinal microglial cells. (a) Microglial cells were transferred into a 16-well chamber slide and stimulated with LPS in the absence of L929 supernatant for 24 h. After stimulation, cells were stained for activation markers, including MHC-II (FITC), CD86 (PE), and CD40 (APC). The slides were observed by confocal microscopy. (b) Microglial cells were transferred into a 16-well chamber slide, incubated with POS-FITC at the ratio of cells to POS-FITC 1 : 5 for 18 h, and stained for CD11b (APC). (c) Microglial cells (1 × 104/well) were transferred into a 96-well plate and stimulated with 1 μg/mL of LPS for 24 h. The supernatant was collected and measured for the presence of IL-1β, IL-12, IL-10, CCL2, GM-CSF, IL-6, TNF-α, and CCL5 using CBA. *P ≤ 0.05 compared with control nonstimulated cell supernatant. Mean ± SEM, n = 3.
Mentions: To characterise the cells, the retinal cultures were transferred into a chamber slide and stained for the surface markers including CD11b, F4/80, and CD45. None of the above markers are specific to the retinal microglial cells. However, microglia are generally characterised as CD11bhigh and CD45low. The majority (>98%) of the cultured cells were positive for CD11b indicating that the cells belong to the mononuclear phagocytic system. The cells expressed high levels of F4/80 (Figure 5(b)) and CD11b (Figure 5(c)) and low levels of CD45 (Figure 5(d)). The cultured retinal microglial cells were negative for MHC-II, CD86, and CD40 (Figures 5(e), 5(f), and 5(g), resp.) indicating that they were not activated. However, microglial cells were positive for MHC-II, CD86, and CD40 in response to LPS stimulation (Figure 6(a)).

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