Revisiting Primary Microglia Isolation Protocol: An Improved Method for Microglia Extraction.

Microglia, the resident immune cells of the central nervous system, play a crucial role in maintaining neural homeostasis and in regulating neurodevelopment, neuroinflammation, tissue repair, and neurotoxicity. They are also key contributors to the pathogenesis of various neurodegenerative disorders, underscoring the need for in vitro models that accurately recapitulate disease-relevant conditions. Among the available isolation methods, the classical mixed glial culture shaking technique remains the most commonly employed, while alternatives such as magnetic bead separation and fluorescence-activated cell sorting (FACS) offer higher purity but are often constrained by technical complexity and cost. In this study, we refined the traditional shaking method by supplementing specific cytokines during culture to enhance microglial viability and proliferation. Our optimized protocol produced primary microglia with higher purity, greater yield, and improved viability compared with the conventional approach, thereby increasing experimental efficiency while substantially reducing time, animal usage, and overall cost. Key features • The microglial cells obtained using this protocol achieve a purity of approximately 90%. • This protocol maximizes the viability of primary microglial cells. • The entire procedure requires a minimum of 9 days to complete. • Antibiotic or antifungal solutions are not used in this protocol.