Microglia are the resident immune cells of the CNS and are hypothesized to influence aging in the brain. Like somatic cells, microglia can be replaced by self-renewal. Recently, some studies have suggested that new microglia derive from asymmetric cell division of a progenitor population. Microglia progenitor cells have been difficult to study due to a lack of specific molecular markers of this population. However, the Garden lab has recently identified novel candidate markers. We hypothesize that in neurodegenerative disorders associated with advanced age, microglia progenitor senescence may contribute to disease pathology. To efficiently study the senescence of microglia progenitors, we turned to neonatal mixed glia cultures, in which the presence of microglia progenitors has long been inferred. In these cultures, microglia are harvested from cells floating above a monolayer culture of mixed neonatal glial cells. The size of each microglia harvest generally decreases with successive harvests. This suggests that microglia progenitors in the attached monolayer may become senescent after multiple rounds of the cell cycle, leading to stagnation in the generation of new floating microglia. We evaluated microglia progenitor senescence in neonatal mixed-glia cultures by labeling with BrdU, a thymidine analog taken up by proliferating cells and remaining in their daughters. Microglia harvested from these cultures weekly were assessed for BrdU incorporation using flow cytometry and immunofluorescent microscopy. We co-labeled floating microglia and dissociated monolayer mixed glia cultures with antibodies directed against a progenitor marker (CD133), a microglia marker (Iba1), and BrdU. The attached mixed-glia cell layer was also labeled for SA-ß-Gal, an indicator of cellular senescence. Progenitor senescence will be detected by a decrease in CD133/BrdU-positive cells and an increase in CD133/ SA-ß-Gal positive cells.