Apc conjugated anti cd206

For surface marker staining, cells were stained in PBS supplemented with 1% FBS for 30 min on ice with the following antibodies (PerCP‐conjugated anti‐CD3, BV421‐conjugated anti‐CD4, PE‐conjugated anti‐CD8, BV570‐conjugated anti‐CD45, BV421‐conjugated anti‐F4/80, BV650‐conjugated anti‐CD86, APC‐conjugated anti‐CD206, PE‐Cy7‐conjugated anti‐CD25, FITC‐conjugated anti‐γδ TCR, APC‐Cy7‐conjugated anti‐CD11b, PE‐conjugated anti‐Ly‐6G, FITC‐conjugated anti‐CD49b, PE‐conjugated anti‐CD19, PE‐conjugated anti‐ST2 antibodies, and APC‐conjugated anti‐NKp46: BD Biosciences, San Jose, CA, USA). After fixation and permeabilization, staining with an APC‐conjugated anti‐FOXP3 antibody (eBioscience) was performed according to the manufacturer's instructions. For IL‐33 staining of primary mouse hepatocytes, cells were stained using a biotinylated anti‐IL‐33 monoclonal antibody (Enzo Life Biosciences, Raamsdonksveer, The Netherlands) and PE‐Cy7‐labeled streptavidin (BD Pharmingen, San Diego, CA, USA). All the flow cytometric data were analyzed and plotted using FlowJo software (TreeStar, Ashland, OR, USA).

Macrophages were stained with a Near-IR LIVE/DEAD fixable dead cell stain (Invitrogen) and the following antibodies in three independent cocktails: FITC-conjugated anti-CD16 (Clone CB16, ebioscience), PE-conjugated anti-MerTK (Clone 108928, R&D Systems), APC-conjugated anti-CD206 (Clone 19.2, BD Biosciences), V450-conjugated anti-CD86 (Clone FUN-1, BD Biosciences), FITC-conjugated anti-CD32 (Clone FL18.26, BD Biosciences), PE-conjugated anti-SIRPα (Clone 15–414, AbSerotec), APC-conjugated anti-CD200R (Clone OX-108, Biolegend), eFluor450-conjugated anti-HLA-DR (Clone L243, ebioscience), PE-conjugated anti-CD80 (Clone 2D10, ebioscience), APC-conjugated anti-CD163 (Clone GH1/61, ebioscience), PerCP-conjugated anti-CD14 (Clone MoP9, BD Biosciences) and V450-conjugated anti-CD64 (Clone 10.1, BD Biosciences). Cells were washed and resuspended in stabilizing fixative (BD Biosciences), and data was acquired on a FACSVerse flow cytometer using identical Tube Settings to allow for a comparison of fluorescence values across experiments and donors. Macrophages were identified as live, single cells. Doublets were excluded by SSC-W and SSC-H discrimination. The expression level of each of the markers was expressed as fold changes relative to M(-) or non-repolarized M(-), M(IL-4) and M(HAGG+IL-1β) using GraphPad Prism software.

MDMs were pretreated with FITC-conjugated anti-CD68, BV421-conjugated anti-CD80 (Biolegend, San Diego, CA, USA), APC-conjugated anti-CD163 (BD Biosciences, San Jose, CA, USA), and APC-conjugated anti-CD206 (BD Biosciences, San Jose, CA, USA). Single-cell suspensions were subjected to flow cytometry analysis (FACS Canto^TM II; BD Biosciences). Data were analyzed using the FlowJo software version 10 (FlowJo, Ashland, OR, USA). Events were gated on live and single-cell populations. CD68⁺-cells were gated as macrophages. The percentage of CD80⁺CD163^—, CD80⁺CD206^—, CD80^-CD163⁺-, and CD80^-CD206⁺-cells among these cells was further analyzed. Appropriate fluorescently labeled isotype-matched antibodies (Abs) to irrelevant antigens were used as controls for all analyses.