Time-Resolved Basophil Activation Tests

Basophil activation tests were performed as reported in Santos et al. (2014) (link) at weeks 0, 4, 12, 16, 52, and 104. First, we performed dose-finding experiments in 10 HDM-allergic patients and 5 non-atopic controls. Heparinized whole blood (100 μl) was stimulated for 30 min at 37°C with Der p extract (ALK-Abello A/S, Horsholm, Denmark) diluted in phosphate-buffered saline (PBS, Sigma Diagnostics, St. Louis) at serial sixfold dilutions (150, 15, 1.5, 1.5 × 10^–1, 1.5 × 10^–2, 1.5 × 10^–3 μg/ml). The two allergen concentrations that evoked maximal (i.e., 15 μg/ml) and submaximal (i.e., 0.15 μg/ml) cell stimulation were chosen for the study to investigate the time course of BAT during the SCIT. Before erythrocyte lysis, cells were stained with CD123-PE-Cy5 (BD), CD203c-PE, HLA-DR-ECD, and CD63-FITC (Beckman Coulter). Basophils gated as SSC^low/CD203c⁺/CD123⁺/HLA-DR^– were detected by flow cytometry (Beckman Coulter Epics XL-MCL, United States) and analyzed using FCS Express software (version 4).