Novel <i>In Vitro</i> Culture of Microglia from Aged Mice: Implications for the Future of Aging Neurobiology Research

Aging is associated with elevated levels of inflammation across tissues, a status recognized as "inflammaging." Within the brain, microglia are the resident phagocytic immune cells that are important in both homeostatic and disease states. Aged microglia are susceptible to processes of "inflammaging," which can include higher expression of baseline levels of inflammatory signals, decline in functional activity, and contribution to neurodegenerative processes. Information about microglial function has been gained using in vitro cell culture methods; however, most studies described previously have used microglia cultured from neonatal mice. More recent studies have used microglia cultured from young adult mice, but those using microglia from aged mice are lacking. Considering the distinct changes that come with aging and the important role of microglia in age-related neurologic disorders, there is a need for reliable protocols for studying aged cells specifically. Here, we describe a method to culture primary microglia from aged mice. Collected brain tissue is digested using enzymatic and mechanical techniques and then cultured in specific medium that supports the continued survival and proliferation of adult and aged microglia. To confirm microglial identity, cultured cells were immunostained for microglia-specific markers and imaged by microscopy and flow cytometry. We also compared the activation status of adult and aged microglia that were cultured versus those that were assessed directly after collection. Microglial cultures can easily be manipulated via genetic modifications or pharmacologic intervention to test specific functions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Culturing primary microglia from adult and aged mice.