Isolation and Characterization of Murine Microglia

Mice were anesthetized with 40 mg/kg of pentobarbital sodium and transcardially perfused with ice-cold PBS. Brain tissues were obtained, and cerebrum was dissected and separated into ipsilateral and contralateral hemispheres. Ipsilateral hemispheres were mechanically dissociated using a razor blade and placed in a 15-ml conical tube with digestion solution [0.6 mg/ml of collagenase D (Sigma)]. Then, the mixture were incubated for 30 min at 37°C. After that, a 70-μm strainer was used to generate a single-cell suspension (BD FALCON). Cells were isolated by centrifugation (30 min, 800 × g at 23°C) using 30–70% Percoll gradient solutions (GE Healthcare) (Agalave et al., 2020 (link)). Isolated cells were washed and resuspended in PBS with 0.01% bovine serum albumin (BSA) and then incubated with indicated anti-mouse antibodies for 30 min at 4°C [rat anti-mouse CD45 perCP (BD Bioscience) and rat anti-mouse CD11b FITC (BD Bioscience)]. The population of microglia (CD45 positive and CD11b positive) was sorted.
In flow cytometry, rat anti-mouse Ly6G PE and rat anti-mouse Ly6C APC (BD Bioscience) antibodies were used and incubated with CD45 perCP and CD11b FITC.