Figure 2

Basophil activation assay performed (1) with or (2) without IgE staining. Buffy coat cells suspended in an IL-3-containing buffer were (A) stimulated with birch allergen or (B) not stimulated (negative control - Glycerol Saline). Subsequently, the cells were stained with (1) IgE-FITC, CD63-PE, CD123-PC5, and HLA-DR/CD3/CD19-APC or (2) IgG1 Mouse Isotype Control-FITC, CD63-PE, CD123-PC5, and HLA-DR/CD3/CD19-APC. Basophills were defined as cells that do not express CD3, CD8, CD14, CD19 or HLA-DR (P2), but express CD123 (P3). Activated basophils were defined as CD63+ basophils (P4). Density plot displaying only P4 is shown for the negative control for both versions of staining (with or without IgE). Percentages of activated basophils are shown in the respective P4 gates. Corrected percentage of activated basophils was calculated by subtracting saline control percentage from allergen stimulated percentage of activated (CD63+) basophils. In this example, the corrected percentage of activated basophils on stimulation with Birch is 37.4% (38.4-1.0%) when anti-IgE was used for basophil staining and is 13.7% (14.2-0.5%) when anti-IgE was not used for basophil staining.