Basophil Activation Assay for Allergy

Whole blood samples from all control and experimental groups were collected via cheek puncture at D63 to conduct a basophil activation test (BAT) according to the method described by Torrero et al. [18 (link)]. Briefly, whole blood was incubated (1.5 h at 37°C) with RPMI-1640 medium (Lonza), αIgE (125 ng/ml, eBioscience, Breda, the Netherlands), or whey (20 μg/ml, DMV International) to activate basophils. After red blood cell lysis (Whole Blood Lysing Reagents, Beckman Coulter, Fullerton, CA, USA), cells were stained with anti-IgE-FITC, anti-CD49b-APC, anti-CD4-PE, and anti-B220-PE (eBioscience) to select the basophil population while excluding T and B cells. Median fluorescence intensity (MFI) of activation marker CD200R-PerCp-eFluor 710 was determined with flow cytometry using a FACS Canto II (BD Biosciences, Alphen a/d Rijn, the Netherlands).