Anti cd115

For intracellular-cytokine staining, single-cell suspended splenocytes were incubated with brefeldin A (eBioscience), followed by an additional 5 h of incubation at 37°C. After surface staining with anti-CD3, anti-CD8, anti-CD11b, anti-CD11c, anti-CD19, anti-CD115, anti-F4/80, anti-Ly6C, anti-Ly6G, anti-MHC-II, and anti-NK1.1 antibodies (all from eBioscience), the cells were fixed with 2% formalin, permeabilized with 0.1% saponin, and stained with anti-TNF antibodies (eBioscience) for 30 min at 4°C. B-cell subsets were detected in single-cell suspensions of splenocytes with anti-CD5, anti-CD19, anti-CD21, anti-CD23, and anti-IgM antibodies (all from eBioscience). BD Calibrite (BD Biosciences, USA) beads were added to the samples before acquisition with a BD LSRFortessa.

Whole blood obtained was collected in EDTA-buffer tubes. Samples were subjected to red-blood-cell lysis for further analysis using flow cytometry. Bone marrow cells were harvested by flushing femurs with Hank’s Medium (Hanks’ Balanced Salt Solution + 0.3 mmol/l EDTA + 0.1% BSA) (Gibco by life technologies). Spleen and lymph nodes were mechanically crushed and passed through a 30 μm cell strainer (Cell-Trics, Partec) using Hank’s Medium and to obtain single cell suspensions. Single cell suspensions were subsequently stained with different antibody cocktails and analyzed using a FACS Canto II, using the FACSDiva software (BD Biosciences). Cell populations were discriminated by the following antibody cocktail: anti-CD45, anti-CD115, anti-Gr1, anti-CD11b, anti-B220, anti-CD3, anti-CD4, anti-CD8 (All eBioscience). Cell populations and marker expression were gated as depicted below using the FlowJo analysis program (Treestar): neutrophils (CD45⁺CD115⁻Gr1^high), classical monocytes (CD45⁺CD115⁺Gr1^high), non-classical monocytes (CD45⁺CD115⁺Gr1^low), B-cells (CD45⁺CD115⁻Gr1⁻B220⁺), CD3⁺T-cells (CD45⁺CD115⁻Gr1⁻CD3⁺), CD4⁺T-cells (CD45⁺CD115⁻Gr1⁻CD3⁺CD4⁺) and CD8⁺T-cells (CD45⁺CD115⁻Gr1⁻CD3⁺CD8⁺).

Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5 min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS buffer (1% BSA, 0.05% NaN₃, in PBS) or full RPMI 1640. Cells in FACS buffer were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with: anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend; 20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated ±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h, 37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8 (20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or Ly6C⁺ cells, whole blood (ethylenediaminetetraacetic acid) was stained with anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend), anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing, and analysis by flow cytometry (Accuri C6).