Fc receptor blocking antibody

For surface marker analysis, macrophages were harvested with enzyme-free cell dissociation buffer (Cat# 13151014; Gibco, Waltham, MA, USA). Cells were resuspended in ice-cold FACS buffer (consisting of PBS and 0.5–1% BSA) at a concentration of 1-5 × 10⁶ cells/mL. To block non-specific binding of the primary antibodies, Fc receptor blocking antibody (Cat# 422301; BioLegend, San Diego, CA, USA) was added to the cell suspension. Cells were incubated with the conjugated primary antibodies or the isotype-matched control in the dark for at least 30 min at 4 °C. Cells were washed three times with ice-cold FACS buffer before analyzing with a FACSAria Fusion flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo (version 10, Ashland, OR).
The following antibodies (all purchased from Invitrogen, Waltham, MA, USA) were used for staining surface markers: CD11b-APC (1:20 dilution; Cat# 17-0118-41), CD14-PE (1:20 dilution; Cat# 12-0149-42), CD16-FITC (1:10 dilution; Cat# 11-0168-41), CD86-PE (1:20 dilution; Cat# 12-0869-41), CD163-APC (1:20 dilution; Cat# 17-1639-41) and isotype controls: mouse-IgG1 kappa-APC (1:40 dilution; Cat# 17-47148-1), mouse-IgG1 kappa-PE (1:40 dilution; Cat# 12-4714-81), mouse-IgG1 kappa-FITC (1:200 dilution; Cat# 11-4714-81), mouse-IgG2b kappa-PE (1:40 dilution; Cat# 12-4732-81).

The following anti-mouse antibodies were from eBioscience: FITC-anti-F4/80, APC-anti-CD80, PE-Cy7-anti-CD86, PE-anti-MHC-II, PE-anti-CD11b, FITC-anti-Gr-1, APC-Cy7-anti-CD3, PerCP-Cyanine5.5-anti-CD4, APC-anti-CD8a, PE-anti-IFN-γ, and FITC-anti-IL-17. For intracellular cytokine staining, cells were stimulated with PMA (50 ng/ml, Enzo Life Sciences) and ionomycin (1 nM, Enzo Life Sciences) in the presence of brefeldin A (1 mg/ml, Enzo Life Sciences) for 4–5 h. Surface staining was performed in FACS buffer (0.5% BSA, 2 mM EDTA, 0.02% sodium azide in PBS) in the presence of Fc receptor blocking antibody (BioLegend) for 20 min at 4°C. Cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min at room temperature followed by permeabilization (0.1% BSA, 0.5% saponin in PBS) or with the BD Cytofix/Cytoperm Fixation and Permeabilization Kit. Cytokine staining was performed in permeabilization buffer for 20 min at 4°C. Data acquisition and analysis were performed using BD LSRFortessa X-20 (BD Bioscience) and FlowJo software (Tree Star, Ashland, OR, USA) respectively.

Single-cell suspensions were initially incubated with an Fc receptor blocking antibody (Biolegend, San Diego, CA, USA) and stained with the following antibodies for surface analysis: anti-human CD3 (SK7, BD Biosciences), anti-human CD4 (RPA-T4, BD Biosciences), anti-human CD25 (M-A251, BD Biosciences), anti-human CD127 (HIL-7R-M21, BD Biosciences), anti-human CXCR3 (1C6, BD Biosciences), anti-human CCR4 (L291H4, Biolegend), and anti-human CCR6 (G034E3, Biolegend). PBMCs were stained with anti-CD3 (SK7, BD Biosciences), anti-CD4 (RPA-T4, BD Biosciences), anti-CD25 (M-A251, BD Biosciences) prior to fixation, permeabilization and staining for Foxp3 (259D, Biolegend). For analysis of ST2⁺ Tregs, PBMCs were stained for CD4 and ST2 (RMST2-33, Invitrogen, Carlsbad, CA, USA) prior to staining for Foxp3. For analysis of α-smooth muscle actin (α-SMA)⁺ OFs after coculture with Tregs, OFs were fixed and permeabilized after harvest and stained with an anti-α-SMA antibody (1A4/asm-1, Novus Biologics, Minneapolis, USA).