Fitc anti mouse cd11b antibody

As described (Mylonas et al., 2017 (link)), mouse femur was dissected and bone marrow cells were flushed out with syringe using PBS, after erythrocytes were lysed, the cells were centrifuged at 4°C, 350 g, 5 min to obtain leukocyte precipitation. Then the cells were resuspended in PBS, the percentage of neutrophil was detected by flow cytometry. FITC anti-mouse CD11b antibody (1:50, 101206, Biolegend) and PE anti-mouse Ly6G antibody (1:50, 108408, Biolegend) were used to stain neutrophils and then immediately analyzed with a flow cytometer.

Animal subjects were sacrificed by an overdose of 2% pentobarbital sodium and their brains were carefully extracted and dissected. To dissociate the tissues, a digestion process using Papain (2 mg.ml^− 1, Worthington) in RPMI 1640 medium (Gibico) was carried out at 37℃ for 1 h. The resulting dispersed cells were then filtered using the 70 mm nylon mesh. Subsequently, the resulting cells were further resuspended with a 30% Percoll density gradient (GE Healthcare) and centrifuged at 900 g for 25 min. The cells were isolated by collecting the bottom fraction in the 30% Percoll solution. The samples were then treated with FcR Blocking Reagent (Miltenyi Biotec) for blocking and subjected to flow cytometry analysis for the detection of surface antigens. For fluorescence acquisition, the cells were stained with FITC anti-mouse CD11b Antibody (BioLegend) and PerCp-CyTM5.5 anti-mouse CD45 Antibody (BD Pharmingen). Flow cytometry (FACS Aria II SORP, BD Biosciences, USA) was employed for fluorescence acquisition, and the samples were gated based on CD11b + CD45dim expression, which represents the microglia population.

FITC anti-mouse CD11b antibody (lot: 101206), PE anti-mouse F4/80 antibody (lot: 12310), and APC anti-mouse CD206 antibody (lot: 141708) were all purchased from BioLegend Co.
After the mature BMDMs were treated under different conditions, the cells were collected under 3% BSA in PBS and blocked at room temperature for 15 minutes. The corresponding flow antibody was added at 4°C and incubated away from light for 30 minutes. The antibody was washed with PBS, and a CytoFLEX flow cytometer (Beckman Coulter Inc, USA) was used for sample detection. FlowJo vX.10 software (Ashland, OR, USA) was used for the analysis of FACS data.