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Updated: May 12, 2026
Culturing Microglia from the Neonatal and Adult Central Nervous System
Published on: August 9, 2013
Establishment and characterization of primary adult microglial culture in mice
Hirokazu Ohtaki1, Tomomi Tsumuraya, Dandan Song
1Department of Anatomy, Showa University School of Medicine, Shinagawa-Ku, Tokyo, Japan. taki@med.showa-u.ac.jp
Abstract:
Microglial cells account for approximately 12-15 % of the cells in the central nervous system (CNS). Microglial cells are polarized by pathological stimuli such as cytokines, chemokines, and growth factors, and play important roles in the deterioration and repair of the CNS. Here, we established cultures of primary microglial cells isolated from the brains of adult C57/BL6 mice using Percoll density gradients. The cells were cultured and stained with antibodies against CD11b, glial fibrillary acidic protein, myelin basic protein and NeuN to determine microglial, astroglial, oligodendroglial, and neuronal cells respectively. Moreover, the cells were exposed to interferon-γ (IFNγ) plus interleukin-1β (IL-1β) or IL-4 for 24 h to demonstrate the activating phenotypes with inducible nitric oxide synthase (iNOS), Ym1, and Iba-1 immunoblotting. At least 95 % of the cultured cells were CD11b-positive and -negative for astroglial, neuronal, and oligodendrocyte markers. IFNγ plus IL-1β treatment resulted in classical activation, which was represented by an increase in iNOS. The cells also displayed alternative activation, which increased Ym1 when treated with IL-4. The present study indicates that the microglial cells isolated as described here are a useful tool for elucidating adult microglial function.
Insights
Researchers developed a method to culture primary microglial cells from adult mouse brains. These cells can be activated into distinct phenotypes, offering a valuable tool for studying central nervous system (CNS) repair and deterioration.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglial cells are key immune cells in the central nervous system (CNS).
- They play critical roles in CNS health, disease, repair, and deterioration.
- Understanding microglial cell function is crucial for neurodegenerative disease research.
Purpose of the Study:
- To establish a reliable method for culturing primary adult mouse microglial cells.
- To characterize the activation states of these cultured microglial cells.
- To provide a tool for investigating microglial cell functions in the CNS.
Main Methods:
- Primary microglial cells were isolated from adult C57/BL6 mouse brains using Percoll density gradients.
- Cell purity was confirmed using immunocytochemistry for microglial (CD11b), astroglial, neuronal, and oligodendroglial markers.
- Cells were stimulated with interferon-gamma (IFNγ) plus interleukin-1β (IL-1β) or IL-4 to induce classical or alternative activation phenotypes, respectively.
- Activation markers including inducible nitric oxide synthase (iNOS) and Ym1 were assessed via immunoblotting.
Main Results:
- The isolation method yielded highly pure microglial cultures (≥95% CD11b-positive).
- IFNγ + IL-1β stimulation induced classical activation, marked by increased iNOS expression.
- IL-4 stimulation induced alternative activation, evidenced by increased Ym1 expression.
- Iba-1 immunoblotting confirmed microglial cell presence and response.
Conclusions:
- The described method effectively isolates and purifies primary adult microglial cells.
- These cultured microglial cells exhibit distinct classical and alternative activation states upon stimulation.
- This model system serves as a valuable tool for studying microglial cell biology and function in the CNS.
