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Updated: Jun 6, 2026
Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue
Published on: August 15, 2012
A simple and high-yield method for preparation of rat microglial cultures utilizing Aclar plastic film
Yoshihiro Seki1, Satoshi O Suzuki, Kenta Masui
1Department of Neuropathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Abstract:
Microglia are implicated in both neuroprotection and neurodegeneration, and are a key area of interest with respect to various CNS diseases. Until now, primary microglia prepared by various isolation methods have been widely used to investigate their role in CNS diseases. However, there are some problems with the current isolation methods, such as the numbers of animals required in order to obtain sufficient numbers of microglial cells due to low yields, and also the long periods of culture required. We herein describe a simple, high-yield method for isolating not only primary microglia, but also immortalized microglial cells. Our method allows for the isolation of an almost pure population of microglia with only two steps. First, a primary mixed neural culture was prepared from the brains of 3-day-old postnatal rats. Next, primary microglia were collected for 2 h by adhesion to Aclar plastic film. The average yield by this method was approximately 50 times higher than that of the conventional shaking method. Immortalized microglial cells could also be prepared based on this procedure. A plasmid vector encoding the SV40 large T antigen was transfected into the mixed neural culture using a calcium phosphate precipitation method. Then, proliferating immortalized microglia were collected after several weeks in a similar fashion. Several clones were obtained by limited dilution and one of the immortalized cell lines was designated SMK. The SMK cells exhibited markers specific for the microglia lineage, including Iba-1, CD11b, CD45, CD68, major histocompatibility complex (MHC) class I and MHC class II, but not for the astrocyte-specific markers, GFAP and glutamate aspartate transporter. SMK also showed phagocytic activity. In conclusion, this method resulted in a high-yield preparation of microglial cultures with ease and reproducibility.
Insights
Researchers developed a high-yield method for isolating primary and immortalized microglia, crucial for studying central nervous system diseases. This technique significantly improves cell yield and reduces animal use in microglia research.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Microglia play critical roles in neuroprotection and neurodegeneration in central nervous system (CNS) diseases.
- Current methods for isolating primary microglia are often inefficient, requiring many animals and long culture times.
- There is a need for improved methods to obtain sufficient microglia for research.
Purpose of the Study:
- To develop a simple, high-yield method for isolating primary microglia.
- To establish a reproducible method for generating immortalized microglial cell lines.
- To facilitate research into the role of microglia in CNS diseases.
Main Methods:
- Primary microglia isolation: Two-step process involving primary mixed neural culture from postnatal rat brains and collection via adhesion to Aclar plastic film.
- Immortalized microglia generation: Transfection of mixed neural culture with SV40 large T antigen, followed by collection of proliferating cells and cloning.
Main Results:
- The new method yielded approximately 50 times more primary microglia compared to conventional shaking methods.
- Isolated microglia exhibited specific microglial lineage markers (Iba-1, CD11b, CD45, CD68, MHC class I/II) and phagocytic activity.
- An immortalized microglial cell line, SMK, was successfully established and characterized.
Conclusions:
- The described method offers a simple, high-yield, and reproducible approach for isolating both primary and immortalized microglia.
- This technique can overcome limitations of current methods, reducing animal use and improving efficiency.
- The availability of high-quality microglial cultures, including immortalized lines like SMK, will advance CNS disease research.
