Background

Microglia are the resident immune effector cells of the central nervous system (CNS), endowed with essential functions such as phagocytosis of pathogens and clearance of apoptotic cells. They are primarily responsible for maintaining neural homeostasis, mediating inflammatory responses, and contributing to tissue repair. Increasing evidence has highlighted their pivotal roles in pathological conditions, including neurodegenerative diseases, brain injury, and psychiatric disorders, which have become major focuses of contemporary neuroscience research [1–4]. Although several established in vitro models, such as the murine BV2 microglial cell line, are widely used, they exhibit intrinsic limitations as experimental systems [5]. In detail, the immortalization of BV2 cells may lead to aberrant gene expression, dysregulated signaling pathways, and altered metabolic states. Compared with primary microglia, BV2 cells lack key homeostatic features and immune functionalities, making them less suitable for accurately modeling the physiological responses of microglia in Parkinson’s disease (PD) [6]. Consequently, obtaining primary microglia with both high yield and high purity is of great importance for mechanistic studies in molecular neuroscience. Furthermore, in vitro experiments employing purified primary microglia provide a controllable and physiologically relevant model for dissecting activation pathways and intercellular interactions, thereby facilitating a deeper understanding of their functions under diverse pathological states.

Several approaches have been developed for the isolation of primary microglia from mice, including differential adherence, magnetic-activated cell sorting (MACS), fluorescence-activated cell sorting (FACS), and the mixed glial culture shaking method [7–9]. Among them, MACS and FACS offer high specificity and yield microglia of greater purity; however, these methods are limited by their high cost and relatively low overall recovery. In contrast, differential adherence and shaking methods are technically straightforward and cost-effective for routine use, yet they typically produce cells of lower purity. Therefore, developing an isolation protocol that allows rapid, efficient, and cost-effective acquisition of highly purified primary microglia would be highly valuable for experimental model establishment and subsequent mechanistic studies.

In this protocol, we optimized the traditional mixed glial culture shaking method for microglial isolation. During the initial brain tissue dissociation and early mixed glial culture stage, the neuronal supplement B27 was added to enhance cell survival and preserve microglial function [10,11]. In the subsequent culture phase, macrophage colony-stimulating factor (M-CSF) was supplemented to promote microglial proliferation and expansion [12,13]. This modified protocol not only produced microglia with higher purity but also markedly shortened the isolation timeline, enabling the acquisition of usable primary microglia within as few as 10 days. Moreover, no antibiotic or antifungal agents were used in this protocol. Importantly, the improved method yielded sufficient numbers of more viable cells to support common biochemical analyses—including western blot, polymerase chain reaction (PCR), and proteomic studies—without requiring large numbers of neonatal mice. In addition, the functional competence of the isolated primary microglia was validated through co-culture with lipopolysaccharide (LPS) stimulation (1 μg/mL) [14].