Primary Microglia Isolation from Postnatal Mouse Brains

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 the DMEM. Add 1% Penicillin/Streptomycin to both the medium.
   1. To prepare 20% LADMAC conditioned medium, collect the culture medium from th....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

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