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Updated: Aug 7, 2026
Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures
Published on: July 25, 2022
[A modified protocol of mouse hippocampal primary microglia culture by using manual dissociation, magnetic activated
Ya-Nan Xu1, Li-Jun Zhou1, Ying-Tao Jie1
1Department of Physiology, Zhongshan School of Medicine and Pain Research Center, Sun Yat-sen University, Guangzhou 510080, China.
Abstract:
In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2-4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec's Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-β, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1β and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo.
Insights
This study introduces an improved method for culturing mouse hippocampal microglia, yielding highly pure, active cells resembling those in a healthy brain. The new technique uses magnetic activated cell sorting (MACS) and a specialized TIC medium for better results.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Primary microglia culture is crucial for studying neuroinflammation and brain health.
- Existing methods often yield microglia with altered phenotypes, not reflecting their in vivo quiescent state.
- Developing a reliable method to culture pure, resting-state microglia is essential for accurate research.
Purpose of the Study:
- To optimize the culture method for mouse hippocampal primary microglia.
- To achieve high purity and activity of ramified microglia resembling the in vivo resting state.
- To compare the efficacy of a novel culture medium (TIC) with conventional medium (CM).
Main Methods:
- Hippocampal tissue from young C57BL/6J mice was dissociated into a single-cell suspension.
- Microglia were enriched using two-step magnetic activated cell sorting (MACS) after CD11b immunomagnetic bead incubation.
- Cells were cultured in either complete medium (CM) or TIC medium (serum-free, with TGF-β, IL-34, cholesterol) for 4 days.
Main Results:
- The two-step MACS protocol achieved a high microglia purity of 86.20 ± 0.68%.
- Microglia cultured in TIC medium exhibited quiescent, ramified morphology and maintained low inflammatory gene expression (TNF-α, IL-1β, CCL2).
- Conversely, microglia in CM showed significantly higher inflammatory gene expression after 4 and 7 days.
Conclusions:
- The improved protocol using MACS and TIC medium significantly enhances the yield and purity of mouse hippocampal microglia.
- TIC medium supports the culture of microglia in a quiescent state, closely mimicking their in vivo condition.
- This method provides a more accurate model for studying microglia function in healthy and diseased brain states.
