2.5 Basophil activation test
To prime circulating basophils, whole blood was incubated for 10 mins at
37oC in stimulation buffer (Hepes 20 mM, NaCl 133 mM,
KCl 5 mM, CaCl2 7 mM, CaCl2 3.5 mM, BSA
1 mg/ml, rIL-3 2 ng/ml, Heparin 20 µl/ml, pH 7.4). Basophils were
incubated with RGP extract (0.001 – 1 µg/ml; Stallergenes Greer,
London, UK), BV extract (0.01 – 1 µg/ml; HollisterStier Allergy,
Spokane, WA, US), fluorescent allergen tetramers (0.01, 0.1, 1 µg/ml) or
streptavidin conjugate only (1 µg/ml) for 20 mins at
37oC. In parallel, stimulations with rabbit anti-human
IgE (0.1 – 10 µg/ml; Agilent) or fMLP (8 nM; Sigma, St. Louis, MO, US)
were performed to control for FcεRI-dependent and FcεRI-independent
degranulation, respectively.36 Activation was stopped
by incubating on ice for 5 mins. Cells were washed with cold wash buffer
(Hepes 20 mM, NaCl 133 mM, KCl 5mM, EDTA 0.27 mM, pH 7.3) prior to flow
cytometry.