Efluor 450 anti mouse mhc class 2

For routine verification of purified samples, cells resuspended in FACS buffer (0.1% BSA in PBS) were incubated with 1μg/ml anti-CD16/CD32 (Biolegend, UK, cat # 101301) to block Fc receptors and stained with anti-mouse CD11b-PE (Biolegend, cat # 101207, clone: M1/70), CD45-Pacific Blue (Biolegend, cat # 103125, clone: 30-F11) and F4/80-APC (Biolegend, cat # 123115, clone: BM8). To assess overlap between microglial CD11b and EGFP expression in mixed brain cell suspensions from Csf1r-EGFP mice, samples were stained as above and microglia identified according to their characteristic CD11b⁺F4/80⁺CD45^lo profile. To measure MHC-II expression on microglia from mixed brain cell suspensions, cells were stained with RPE-Alexa Fluor 750 anti-mouse CD11b (AbD Serotec UK, cat # MCA74P750T), APC anti-mouse CD45 (Biolegend, cat # 103111) and eFluor 450 anti-mouse MHC Class II (eBioscience, UK, cat # 48-5321-80). Flow cytometry was performed using a FACS Aria IIIu or LSR Fortessa (Becton Dickenson, UK) and data analysed using FlowJo software (FlowJo, OR, USA).