Anti ccr3

Basophil activation tests were performed as previously described. 28 Briefly, titrated amounts of recombinant allergens or apple extract (0.25-100 ng/mL) in HEPES calcium buffer (pH 7.4) supplemented with IL-3 (2 ng/mL) were added to heparinized whole blood from 4 untreated subjects with BPRAA who displayed rMal d 1-specific IgE levels of 1.1, 1.4, 2.6, and 5.8 kU A /L, respectively. As a negative control, blood was stimulated with HEPES/IL-3 to confirm that basophils were not activated nonspecifically. After incubation for 15 minutes at 378C the reaction was stopped with HEPES/EDTA (20 mmol/L) buffer and cells were stained with anti-CCR3 (allophycocyanin), anti-CD123 (peridinin-chlorophyllprotein), and anti-CD63 (phycoerythrin) (all from BioLegend, San Diego, Calif). After lysis of erythrocytes, basophils were defined as CD123 1 CCR3 1 cells by using BD FACSCanto II and BD FACSDiva software (version 6.1.3) (BD Pharmingen, San Jose, Calif). The concentration of allergen that induced approximately 40% to 50% of CD63 1 basophils in reference to untreated cells was incubated with the indicated pre-and post-SLIT serum samples for 1 hour at 378C before addition to heparinized whole blood of the respective individual.