Anti c3ar af647

Heparinized whole blood was drawn from all study participants. Blood was diluted 1:1 (v:v) with RPMI 1640 (Sigma–Aldrich), added anti‐C3aR‐AF647 (BD), anti‐CD3‐BV711 (BD), anti‐CD14‐APC‐eflour780 (ebioscience), anti‐CD88‐PE‐Cy5 (Biolegend), anti‐CD123‐BV650 (BD), anti‐CD124‐AF700 (R&D) anti‐CD193‐BV421 (BD) and anti‐CD294‐PE‐CF594 (BD) or stained with anti‐CD3‐BV711 (BD), anti‐CD14‐APC‐eflour780 (ebioscience), anti‐CD32‐FITC (BD), anti‐CD123‐BV650 (BD), anti‐CD172a‐PerCP‐eFlour^®710 (ebioscience), anti‐CD193‐BV421 (BD), anti‐CD200R‐PE (Biolegend), anti‐CD300a‐AF647 (Novus) and anti‐Siglec‐8‐AF700 (R&D Systems). Blood was incubated with or without 1 μg/ml polyclonal goat anti‐human IgE (KPL) for 30 min at 37°C using a water bath. Erythrocytes were lysed with BD FACS™ lysing solution (BD). Cells were washed and stained with anti‐FcϵRI‐PE‐Cy7 (Biolegend) for 30 min at 4°C. Cells were washed, fixated using BD CellFix™ (BD) and acquired on a BD Fortessa flow cytometer. Data were analyzed as described above. Basophils were gated as CD3^‐CD14^‐CD193⁺CD123⁺ cells (Fig. S2A).

Resting, IgE‐sensitized and anti‐IgE‐stimulated PBdMC were harvested and stained with Fixable Viability Dye eFluor® 780 (eBioscience, San Diego, CA) for 30 min at 4°C. PBdMC were then washed in PBS (Sigma–Aldrich) and stained with anti‐C3aR‐AF647 (BD Biosciences, Franklin Lakes, NJ), anti‐CD88‐PE‐Cy5 (Biolegend, San Diego, CA), anti‐CD117‐BV650 (BD), anti‐CD124‐AF700 (R&D Systems, Minneapolis, MN), anti‐CD126‐BV421 (BD), anti‐CD203C‐BV510 (BD), anti‐CD218a‐FITC (Biolegend), anti‐FcϵRI‐PE‐Cy7 (Biolegend), anti‐ST2‐PE (R&D Systems), and anti‐TSLPR‐BV605 (BD) or stained with anti‐CD32‐FITC (BD), anti‐CD48‐BV421 (BD), anti‐CD117‐BV650 (BD), anti‐CD172a‐PerCP‐eFlour^®710 (ebioscience), anti‐CD200R‐PE (Biolegend), anti‐CD300a‐AF647 (Novus Biologicals, Littleton, CO), anti‐FcϵRI‐PE‐Cy7 (Biolegend) and anti‐Siglec‐8‐AF700 (R&D Systems) for 30 min at 4°C. Cells were washed and acquired on a BD Fortessa flow cytometer. Data were analyzed with FlowJo software version 10 (TreeStar, Ashland, OR). PBdMC were gated as viable CD117⁺FcϵRI⁺ cells (Fig. S1). Receptor expression was quantified using median fluorescence intensity of the specific stain subtracted that of isotype‐matched control (Δ MFI).