2.4g2 antibody

For intracellular cytokine staining, cells were treated with 1 μL of GolgiPlug^TM (BD Biosciences, USA) according to the manufacturer’s instructions, just before stimulation with LPS. BD Cytofix/Cytoperm^TM kit (BD Biosciences, USA) was used for staining according to the manufacturer’s instructions. At indicated time points, cells were harvested, washed, and incubated with 2.4G2 antibody (BD Biosciences, USA) to block FcγRII/III receptors. Antibodies used in this study: PE-labeled anti-F4/80, biotin-conjugated anti-CD11b, avidin-conjugated PECy5 (BioLegend, USA), PE-labeled anti-MHC class II, biotin-conjugated anti-CD86 (BioLegend, USA), avidin-conjugated ECD (Beckman Coulter, USA), PE-labeled anti-TNFα, PE-labeled anti-IL-6, and biotin-conjugated IL-12p40 and avidin-conjugated PECy5 (BioLegend, USA). The acquired data were analyzed using FlowJo vX (TreeStar, USA).

Markers used to identify monocyte/macrophage subsets included: F4/80-PerCP Cy5.5 or APC (clone BM8; eBioscience & Biolegend), Ly6C-FITC, PE or APC-Cy7 (clone AL-21; BD Biosciences), CCR2-PE or APC (Clone 475301; R&D Systems), MHC class II-PE (clone M5/114.15.2; BD Biosciences), CD11c-APC (clone HL3; BD Biosciences), CD206-PE (C-type Mannose Receptor 1, clone MR5D3; AbD Serotec), CD14-PE (clone Sa2–8; eBioscience), MerTK-APC (clone 108928; R&D Systems) and CD64-PE (clone X54-5/7.1.1; BD Biosciences). Surface FcγRII/III was blocked with 2.4G2 antibody (BD Biosciences). Cells were surface-stained for 30 minutes on ice in staining buffer (1x DPBS/1% FCS). Where indicated, cells were fixed for 30 minutes, treated with 1x permeabilization wash (Cytofix/Cytoperm Kit; BD Biosciences) and intracellularly stained with TNF-α-PE (clone MP6-XT22; BD Biosciences).