Isolation and Immunophenotyping of Microglia

Microglia cells were isolated by mechanical dissociation and Percoll gradient centrifugation as previously described (Verheijden et al., 2015 (link)). For immunological profiling, 1 × 10⁵ cells were incubated for 20 min at 4°C using combinations of CD11b-Pe Cy7 (clone M1/70, Biolegend) with either CD11c-APC (clone HL3, BD Pharmingen), F4/80-APC (clone BM8, eBioscience), CD204-Alexa 647 (clone MR5D3, AbD Serotec), CD206-Alexa 647 (clone 2F8, AbD Serotec) antibodies all at dilutions of 1:200 in a total volume of 200 μl. Flow cytometry was performed on a FACS Verse (BD Biosciences), and data was analyzed with BD FACsuite software (BD Biosciences). Injection and subsequent analysis of Bromodeoxyuridine (BrdU; FITC BrdU Flow Kit 559619, BD Biosciences) in mice by flow cytometric analysis has been described (Verheijden et al., 2015 (link)).