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 microglial subtype in AD and other neurodegenerative diseases4,5,6. This sub-population is proximal to the amyloid β (Aβ) deposition. Genes associated with phagocytosis and lipid metabolism (e.g., Apoe, Tyrobp) were found to be up-regulated in these populations6,7. However, the subcellular processes, extracellular and intracellular signaling pathways that regulate the dynamic molecular profile changes in microglia are not fully understood. Particularly, the mechanism underlying chronic microglial activation in neurodegenerative diseases remains elusive. Therefore, it is crucial to understand microglia-involved cellular mechanisms since microglia responses clearly contribute to brain development and the progression of neurodegeneration.
Although there are a couple of in vivo and in vitro tools to study microglia, there are still some limitations. It is technically difficult to obtain a large enough number of microglial cells with high purity for routinely carrying out experiments, such as western blot, to elucidate intracellular signaling pathways. Primary microglia culture provides an alternative means to study the biology of microglia. The cultured primary microglia can be applied to analyze microglial phagocytic capacity after gene manipulation, and evaluate pro- and anti-inflammatory cytokines production in response to inflammatory stimuli, and other biological aspects to understand their roles in the brain8. Here, we present a new protocol and describe a step-by-step instruction for the isolation and culture of primary microglia from neonatal mouse brain, with emphasis on the steps that are critical for obtaining healthy and pure primary microglial cells.
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
2. Dissection of mouse brain
NOTE: To evaluate the efficacy of the protocol, here we used CX3CR1GFP knock-in mice for tracing microglia9. CX3CR1GFP/GFP mice and wild type C57BL/6J females at 2- to 8-month were crossed to generate CX3CR1+/GFP mice, both were originally obtained from commercial sources. Mice were housed under a 12-12 h light-dark cycle in a specific pathogen-free (SPF) environment at Shanghai Jiao Tong University. Pups from postnatal day 1 to day 4 can be used for this protocol.
3. Seed the mixed primary cells
4. Collection of primary microglia
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, indicating the isolated microglia are highly pure10. Besides, to further characterize the function of the primary microglia cell in vitro, the isolated microglia were treated with 10 ng/mL and 100 ng/mL LPS for 24 h (as shown in Figure 2). Compared to control, LPS exposure induced an activated phenotype, including upregulating expression of CD68 and transforming to an amoeboid morphology with long and branched processes. The findings suggested the isolated primary microglia cells in this protocol allow for at least successful inflammation investigation.
Figure 1: Cells isolated from p2 mouse pups are of high purity. Representative images showed the purity of primary microglia collected from Day 7 (A) and Day 35 (B) after mixed cell culture. Cells are stained with IBA1 (red), S100β (purple), CC1 (purple). Scale bar 50 µm. Please click here to view a larger version of this figure.
Figure 2: Isolated primary microglia responds well to LPS exposure. Representative confocal images showed the expression of CD68 (gray) and IBA1 (red) at isolated microglia cells after LPS treatment. Scale bar 50 µm. Please click here to view a larger version of this figure.
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 cross-contamination. Second, the tissue dissection and seeding steps should take an experienced researcher about 1.5-2 h for 10-12 pups. Large-scale preparation is feasible but definitely extends the time, especially the time for tissue dissection. However, it is better to finish the cell isolation in a timely fashion to reduce cell exposure to damage signals, so as to minimize hypoxic and ischemic induced cell activation. Third, it is necessary to gently handle the mixed cell culture to avoid disruption of astrocytic monolayers. Additionally, do not vigorously shake the flask during the isolation process to minimize excessive microglial activation.
In the subsequent collection steps, previous protocols highlight that mechanical dissociation can increase the yield of microglia. For instance, Tamashiro et al. recommended shaking the culture flasks for 1 h at 100 rpm to enhance microglial detachment from the astrocytic layer12. Lian et al. suggested that it was necessary to tap the flasks when harvesting the cells11. However, consideration should be taken into account that although the yield of microglia could be increased by introducing mechanical force, additional mechanical manipulation also increases the dissociation of oligodendrocytes and astrocytes, leading to a reduction of cell purity. Moreover, shaking and vigorously tapping often generate lots of air-bubbles to the culture medium, resulting in a higher risk of contamination. Also, additional time will be needed to remove the bubbles and minimize cell contamination.
In this protocol, we were able to isolate healthy microglia from Day 40 mixed cultured cells, which were still suitable for live-cell images to record microglial morphology and function as we showed in the video. However, It should be noted that the properties of microglia isolated from different time points might differ. Caldeira et al. found that primary microglia show an irresponsive and senescent phenotype when cultured in vitro for 16 days13. Therefore, it is important to assess the cell state of primary microglia to minimize the artifact introduced by in vitro culture system. It is highly recommended that sequential experiments should be conducted with primary microglial cells that are isolated at the same timepoint, and triplicate experiments should be performed on microglia derived from independent isolation.
The authors have nothing to disclose.
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 (Grant No. JCYJ20180507182033219 and JCYJ20170818163320865) (B.P.) and (Grant No. JCYJ20170818161734072) (S.X.).
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 |
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