Figure S1. Panel A (n=3); kinetics of PMA-stimulated CD63 and CD203c expression. Basophils were stimulated with 50 ng/mL PMA and expression of CD63 and CD203c assessed by flow cytometry, (▪) CD63, (○) CD203c.

Figure S2. Example of images of degranulation sac formation during stimulation of basophils with FMLP. The image series follows a small group of cells labelled with fura-2. The sequence flows from left to right and top to bottom with intervals of approximately 10 s, the upper left representing 10 s after FMLP addition.

Figure S3. Relationship between the cytosolic calcium response and degranulation sac formation at the single cell level. The plot shows results from one preparation of basophils, labelled with fura-2 and stimulated with anti-IgE Ab @ 0.5 μg/mL. Degranulation sacs and the cytosolic calcium response for individual cells were measured as described in the methods. Each symbol represents the results for a single cell. For this particular plot, the Spearman rank correlation was 0.550.

Figure S4. Cytosolic calcium response in basophils with and without PKC inhibitor Ro-31-8220. Basophils were pre-incubated with vehicle control (DMSO) or Ro-31-8220 at 3 μm for 10 min prior to stimulation with anti-IgE Ab at 0.5 μg/mL. The traces represent the average of 3 experiments; for the control response (○), the gray region surrounding the primary trace represents the ±SEM for the averaged kinetic curve. For the response in the presence of Ro-31-8220 (•), the error bars represent ±SEM.

Table S1. Survey of the characteristics of the human basophil response under various conditions of activation. For the purposes of comparison, the three dominant methods of assessing basophil activation, histamine release, CD63 and CD203c expression, are examined as three groups, group I = histamine release, group II = CD63 expression, group III = CD203c expression. In other studies, group II and group III may contain other expression markers such as CD107a, CD107b, CD13, CD69, and CD164. In this table, there are 4 major categories of the response that differentiate the three compartments from each other. These categories are highlighted with different levels of gray background. * = determined in this study, § = normally IL-3 does not induce histamine release but it may in selected patients (see reference (8)), °‡ = supra-optimal polyclonal anti-IgE has unique characteristics, see text.

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