Flow Cytometry Microglia Immunophenotyping

Flow cytometry analysis of immunostained cells was performed following standard cell protocols. Prior to antibody labelling, the cell suspensions were incubated with anti-murine CD16/CD32 FC-Receptor (1:100, Cat. #14-0161-81, eBioscience, CA, USA) blocking reagent at 4 °C for 10 min. After blocking, the microglia were stained with FITC-conjugated mouse anti-rat CD11b (1:100, Cat. #554982, BD Biosciences, USA) and PerCP/Cy5.5-conjugated anti-rat CD45 (1:50, Cat. #202220, Biolegend, CA, USA). The microglia were then fixed and permeabilized with BD fixation/permeabilization solution (Cat. #554714, BD Cytofix/Cytoperm™, USA) for 20 min. The microglia were washed with BD Perm/Wash buffer (Cat. #554714, BD Cytofix/Cytoperm™, USA), resuspended in BD Perm/Wash buffer, and incubated with anti-iNOS (1:100, Cat. #ab15323, Abcam, UK) and rabbit mAb anti-arginase-1 (Arg-1) (1:100, Cat. # 3668s, Cell Signaling Technology, USA) primary antibodies for 30 min followed by a PE-conjugated anti-rabbit IgG (H+L) secondary antibody (1:100, Cat. #8885s, Cell Signaling Technology, USA). The cells were analyzed using a CytoFLEX instrument (Beckman Coulter Biotechnology, SuZhou). Ten thousand events were recorded, and microglia were identified by CD11b⁺/CD45^low expression [33 (link)]. The results were analyzed using CytExpert software (Beckman Coulter Biotechnology, SuZhou).