Isolation of Rodent Microglia to Assess Gene Silencing and Drug Targeting

Aging is the strongest risk factor for neurodegeneration, with a majority of the elderly population experiencing cognitive decline. While many factors drive disease progression, impaired microglial dysfunction has recently emerged as a common hallmark of many age-associated neurodegenerative disorders. As the resident immune cell of the central nervous system (CNS), microglia serve important functions including monitoring the CNS for pathogens and apoptotic signals. However, in disease states, chronic overactivation of microglia contributes to neurotoxicity and promotes disease progression, particularly in the context of Alzheimer’s Disease. The potential to explore microglia immunomodulation in culture is an invaluable tool to help mitigate age-associated neurodegenerative disease. We established a protocol to isolate microglia from the rodent CNS and maintain them in culture. This efficient method involves dissociating mouse brains into single cells with high viability, followed by magnetic sorting using beads coated with the antibody cd11b, a microglia-specific marker. Microglial identity was confirmed by flow cytometry for cd11b and immunostaining for iba1. Microglia were maintained in culture for up to a week and retained high viability and cellular identity. Furthermore, the magnetically sorted microglia were highly abundant with over 6,000 cells and enriched in microglia-specific markers compared to the remainder of the dissociated cell population. To demonstrate the versatility of the protocol, microglia were similarly isolated from rat brains and successfully cultured. For future work, observing phenotypic changes in microglia after siRNA-mediated gene silencing and applying therapeutics to diseased murine microglia will be explored in hopes of improving our understanding of treatments for neurodegenerative disease.