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Protocol

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Neuroscience

Primary Microglia Isolation from Postnatal Mouse Brains

Published: February 25th, 2021

DOI:

10.3791/62237

1Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, 2Division of Biology and Biomedical Science, Washington University in St. Louis, 3Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 4Department of Neurosurgery, Huashan Hospital, Institute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Fudan University, 5Co-innovation Center of Neuroregeneration, Nantong University
* These authors contributed equally

Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.

Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations in vitro.

Microglia, the resident immune cells in the central nervous system (CNS), play pivotal roles in the maintenance of homeostasis, which respond to the neuropathological challenges1. Recently, intensive investigations have been conducted to figure out the physiological functions of microglia, e.g., in Alzheimer's disease2. Currently, the transcriptional profile of microglia at the single-cell resolution obtained during the CNS development, aging, and disease provides a better understanding of microglial function in healthy and diseased brains3. Previous studies identified a disease-associated mic....

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All the animal procedures were approved by the committee on Animal Research and the Institutional administration panel of laboratory animal care at Shanghai Jiao Tong University. Every effort has been made to minimize animal suffering.

1. Preparation of culture buffer, flasks, and coverslips

  1. Culture medium and microglia culture medium: Supplement DMEM with 10% FBS to make the culture medium. The microglia culture medium is made by adding 10% FBS and 20% LADMAC conditioned medium to.......

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Primary microglia collected from CX3CR1+/GFP at Day 7 and Day 35 after cell seeding were demonstrated in Figure 1. As shown in the immunofluorescence staining of microglia/macrophage cell marker IBA1, all IBA1 positive cells are positive for the GFP signals, and negative for S100β (astrocyte marker) and CC1(oligodendrocyte marker), suggesting that the purified GFP positive cells are indeed microglia. Next, it was found over 95% of isolated cells are GFP positive cells, i.......

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This protocol is based on the methods described previously with some modifications11. Tips for improving the viability and purity of isolated microglia are listed as follows. First, take care to avoid contamination when preparing buffers used for tissue isolation and cell culture. Make sure the surgery tools, containers, and plastic equipment are sterile. Usually, we perform the brain dissection in a separate tissue culture hood aside from the cell culture hood for general cell culture to avoid cr.......

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The authors would like to show their respects to the heroes combating the COVID-19 outbreak. The authors thank Fang Lei (Fudan University) for the excellent laboratory management, Dr. Zikai Zhou, Dr. Jing Li, and Dr. Guiqing He (Shanghai Mental Health Center) for the discussion of microglia isolation. Last but not least, the authors show their gratitude and respect to all animals sacrificed in this study. This study was supported by the National Key R&D Program of China (Grant No. 2017YFC0111202) (B.P.), National Natural Science Foundation of China (Grant No. 31922027) (B.P.) and (Grant No. 32000678) (Y.R.), and Shenzhen Science and Technology Research Program (Gr....

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Name Company Catalog Number Comments
Cell strainers, 40 µm ThermoFisher Scientific 22-363-547
DNase I  Sigma 11284932001
Dulbecco's Modified Eagle Medium (DMEM) Gibco by Life Technologies C11995500BT
Dulbecco's Phosphate Buffered Saline (DPBS) Gibco by Life Technologies 14190-144
Fetal Bovine Serum (FBS) Gibco by Life Technologies 10099141
Papain, Suspension Sangon Biotech Papain, Suspension
Penicillin-Streptomycin 100X solution Hyclone SV30010
Poly-D-Lysine ThermoFisher Scientific A3890401

  1. Prinz, M., Jung, S., Priller, J. Microglia biology: one century of evolving concepts. Cell. 179 (2), 292-311 (2019).
  2. Perry, V. H., Nicoll, J. A., Holmes, C. Microglia in neurodegenerative disease. Nature Reviews. Neurology. 6 (4), 193-201 (2010).
  3. Parker, K. R., et al. Single-cell analyses identify brain mural cells expressing CD19 as potential off-tumor targets for CAR-T immunotherapies. Cell. 183 (1), 126-142 (2020).
  4. Tay, T. L., Dautzenberg, J. S., Grun, D., Prinz, M. Unique microglia recovery population revealed by single-cell RNAseq following neurodegeneration. Acta Neuropathologica Communications. 6 (1), 87 (2018).
  5. Deczkowska, A., et al. Disease-associated microglia: a universal immune sensor of neurodegeneration. Cell. 173 (5), 1073-1081 (2018).
  6. Keren-Shaul, H., et al. A unique microglia type associated with restricting development of Alzheimer's disease. Cell. 169 (7), 1276-1290 (2017).
  7. Krishnaswami, S. R., et al. Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nature Protocols. 11 (3), 499-524 (2016).
  8. Timmerman, R., Burm, S. M., Bajramovic, J. J. An overview of in vitro methods to study microglia. Frontiers in Cellular Neuroscience. 12, 242 (2018).
  9. Huang, Y., et al. Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion. Nature Neuroscience. 21 (4), 530-540 (2018).
  10. Garcia, J. A., Cardona, S. M., Cardona, A. E. Analyses of microglia effector function using CX3CR1-GFP knock-in mice. Methods in Molecular Biology. 1041, 307-317 (2013).
  11. Lian, H., Roy, E., Zheng, H. Protocol for primary microglial culture preparation. Bio-protocol. 6 (21), (2016).
  12. Tamashiro, T. T., Dalgard, C. L., Byrnes, K. R. Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue. Journal of Visualized Experiments: JoVE. (66), e3814 (2012).
  13. Caldeira, C., et al. Microglia change from a reactive to an age-like phenotype with the time in culture. Frontiers in Cellular Neuroscience. 8, 152 (2014).

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