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Updated: Apr 27, 2026
Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium
Published on: March 9, 2018
Culture and characterization of microglia from the adult murine retina
Gayathri Devarajan1, Mei Chen2, Elizabeth Muckersie1
1Infection and Immunity, School of Medicine, University of Aberdeen, Foresterhill AB25 2ZD, UK.
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.
Insights
Researchers developed a simple protocol to isolate and culture adult mouse retinal microglia. The method optimizes cell density and culture medium for high yield and purity, enabling functional studies of these important immune cells.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia are the primary immune cells of the central nervous system, playing crucial roles in retinal health and disease.
- Establishing reliable methods for isolating and culturing adult retinal microglia is essential for studying their function in vitro.
Purpose of the Study:
- To establish and optimize a protocol for the isolation and culture of murine adult retinal microglia.
- To characterize the phenotype and functional responses of these cultured cells.
Main Methods:
- Murine adult retinal single-cell suspensions were prepared and cultured under optimized conditions (medium, growth factors, cell density).
- Microglia purification from mixed cultures was performed.
- Phenotypic characterization using surface markers (CD45, CD11b, F4/80) and functional assessment via lipopolysaccharide (LPS) stimulation and cytokine secretion (cytometric bead array) were conducted.
Main Results:
- Optimized culture conditions (0.75 × 10^6 cells/cm^2, DMEM/F12, 20% FCS, 20% L929 supernatant) yielded high-purity microglia by day 10 (over 98% CD45+, CD11b+, F4/80+).
- LPS stimulation induced secretion of proinflammatory cytokines (IL-6, TNF-α) and expression of activation markers (CD86, CD40, MHC-II).
Conclusions:
- A straightforward and effective method for isolating and culturing adult mouse retinal microglia has been successfully developed.
- The cultured microglia exhibit characteristic phenotypes and functional responses, making them suitable for further research into retinal immunity and disease models.
