Basophil activation induced by antibody 2.4G2 and 2.4G2 scFv-MSA. (A) To analyze CD200R3 levels on basophils, RBCs in peripheral blood were lysed by ammonium chloride buffer before staining with anti-CD49b-Pacific Blue, anti-FcεRIα-PerCP/Cy5.5, and anti-CD200R3-FITC. The population within gate P1 represents PBMCs (left) and was further gated based on CD49b and FcεRIα expression levels. The population within P2 (middle) represents basophils (P2 shown in FCS and SSC plot, right), evidenced by expression of CD200R3 (B). (C) Mice were bled before treatment and 4 and 24 hours after administration of 0.43 nmol (65 µg) 2.4G2 or equimolar amounts of 2.4G2 scFv-MSA or HSA. Samples were stained with anti-CD49b-Pacific Blue, anti-FcεRIα-PerCP/Cy5.5, and anti-CD200R3-FITC. All samples were analyzed by MACS Quant. Data were analyzed by Flowjo V10 software. Dot plots and histograms representative of 6 to 7 mice per group from 4 independent experiments.