Functional Cytometric Tests
All the assays were done with Af, Penicillium notatum (Pen), and Alternaria alternata (Alt) extracts (Bühlmann Laboratories®, Schönenbuch, Switzerland). BAT was performed with the Flow2CAST method (Bühlmann Laboratories®), using CCR3 (CD193) and CD63 as basophil identification and activation markers, following the manufacturer's instructions. Positive controls were anti-RFcεI and the bacterial peptide fMLP. For LST, whole blood was incubated in a 96-well plate with RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) and sequential allergen dilution for 24 h under 5% CO2. Phytohemagglutinin (PHA, Thermo Fisher Scientific), 10 μg/L, was used as a positive control. Each well was harvested and stained with a mix of the following antibodies: PerCP-anti-CD45 (clone 2D1), FITC-anti-CD3 (clone SK7), APC-anti-CD4 (clone SK3), PE-anti-CD8 (clone SK1), and PeCy7-anti-CD69 (clone L78) (BD Biosciences®, San Diego, California). Flow cytometry was performed on a FACS Canto II (Becton Dickinson, Le Pont de Claix, France) and at least 200 basophils per sample were analyzed for BAT and 10,000 lymphocytes for LST.