Basophil Identification and Stimulation Protocol

Whole blood was collected in a heparin and an EDTA tube. The EDTA tube was used for external CBC analysis. The heparin tube was kept at 18–25°C. Basophil identification was done using unstimulated blood (PBS) as well as blood stimulated with either Anti-IgE-FITC (Thermo Fisher, Waltham, MA) or fMLP (Sigma, St. Louis, MO). The samples were incubated for 20 min at 37°C followed by 10 min at 4°C (4 (link), 14 (link), 21 (link)–24 (link)). Each sample was stained with the following antibodies Anti-IgE-FITC (Clone Ige21), anti-CD193-PE (Clone 5E8), anti-CD123-PerCPCy5.5 (Clone 6H6), anti-CD203c-PECY7 (Clone NP4D6), anti-CRTH2-APC (Clone BM16), anti-CD3-AF700 (Clone UCHT1), anti-CD45-EF506 (Clone HI30) anti-FcɛRI-SB600 (Clone AER-37) (all Thermo Fisher, Waltham, MA) and anti-HLADR-Pacific blue (L243) (Biolegend, San Diego, CA) for 30 min at 4°C. Each antibody was titrated to obtain the best separation (25 (link)). The red blood cells were lysed using BD FACS lysis solutions (BD Bioscience, San Jose, CA) and resuspended in 400 μl PBS before acquisition.