Anti cd16 32 antibody clone 2.4g2

A single cell suspension was obtained by chopping the tissue and then forcing the dissociated CNS tissue through a 70 μm cell strainer (Falcon, United States) in HBSS supplemented with 2% fetal bovine serum (FBS). Myelin clearance was obtained by centrifugation on 37% Percoll (GE Healthcare Bio-Science AB) followed by aspiration of the myelin layer. Cells were incubated in blocking solution containing HBSS, 2% FBS, anti-CD16/32 antibody (Clone 2.4G2, BD Biosciences), Syrian hamster IgG (50 μg/ml, Jackson ImmunoResearch Laboratories Inc.) and 0.01% sodium azide, and then labeled with fluorophore-conjugated antibodies (BioLegend): anti-CD45 (clone 30-F11), CD11b (M1/70), F4/80 (BM8), GR-1 (RB6-8C5), NK1.1 (PK136), CD4 (GK1.5), and TCRβ (H57-597). Fluorescence data were acquired on an LSRII flow cytometer (BD Biosciences) with FACSDiva software (BD Biosciences) and analyzed with Flowlogic (Inivai Technologies).

Immunostaining was performed as previously described on single cell suspensions derived from murine aortas to characterize immune cells [15 ]. To block Fc receptors, an unconjugated anti-CD16/32 antibody (clone 2.4G2, BD Bioscience) was used for mouse samples. Living cells were selected using Fixable Viability Dye-eFluor™ 780 (1:2000, eBioscience) and different cell populations were defined using anti-mouse fluorochrome-conjugated antibodies (Supplementary Table S1). Antibody staining of transcription factors and cytokines was performed using transcription factor fixation/permeabilization concentrate and diluent solutions and cytofix/permeabilization solutions, respectively (BD Biosciences). Flow cytometry analysis was performed on a Cytoflex S (Beckman Coulter) and the acquired data were analyzed using FlowJo software (version 10.7).

In vitro polarized macrophages, treated with 3GA-494 or 3GA-ctrl, were analyzed with flow cytometry to determine macrophage polarization phenotypes. Fc receptors were blocked using TruStain FcX (Biolegend) and an unconjugated anti-CD16/32 antibody (clone 2.4G2, BD Bioscience) for human and murine samples, respectively. Living cells were selected using Fixable Viability Dye eFluor 780 (1:2,000, eBioscience), and different cell populations were defined using anti-human and anti-mouse fluorochrome-conjugated antibodies. In human macrophages, the number of proinflammatory macrophages (CCR7⁺CD86^hi) or anti-inflammatory macrophages (CD206⁺) was quantified and shown as a percentage of positive cells within live CD45⁺CD11b⁺ cells. For murine macrophages, intracellular iNOS and Arg1 were stained using transcription factor fixation/permeabilization concentrate and diluent solutions (BD Biosciences). The number of proinflammatory macrophages (iNOS⁺) or anti-inflammatory macrophages (Arg1⁺) was quantified and shown as a percentage of positive cells within live CD11b⁺F4/80⁺ cells. MFI per cell was also quantified. Fluorescence-activated cell sorting analysis was performed on a Cytoflex S (Beckman Coulter), and the acquired data were analyzed using FlowJo software.