Biologic effects of the cross-linking of FcεRI on ROSA^{KIT WT} cells. ROSA^{KIT WT} cells were primed first with recombinant human IL-4 (recombinant human IL-4; 20 ng/mL) and human monoclonal IgE (2 μg/mL) for 5 days to increase the expression of FcεRI. (A) To determine CD203c upregulation, ROSA^{KIT WT} primed cells were stimulated by anti-human IgE (5 µg/mL) or by Ca-ionophore (Cai) (1 µM) for 1 hour. After labeling with an anti-human CD203c Ab coupled with allophycocyanin (APC), the fluorescence intensity was measured by using a FACSCalibur flow cytometer. (B) To determine β-hexosaminidase release, ROSA^{KIT WT} primed cells were stimulated by anti-human IgE or by Cai for 1 hour. β-hexosaminidase enzymatic activity was measured in the cell-free supernatants and after cell lysis as described in Materials and methods. (C) To determine histamine release, primed ROSA^{KIT WT} cells were stimulated with various concentrations of anti-human anti-IgE Ab at 37°C for 30 minutes. Thereafter, cells were centrifuged at 4°C, and the supernatants and cell pellets were analyzed for histamine content by using a specific radioimmunoassay kit. Results of histamine release are expressed as percentage of total histamine. (D) To examine synthesis of PGD2, primed cells were stimulated by anti-human IgE (5 or 10 µg/mL) or by Cai (1 µM) for 1 hour. Thereafter, cells were centrifuged, and the cell-free supernatants were analyzed for PGD2 content by using a specific enzyme immunoassay (EIA) kit. (E) To study the synthesis of TNF-α, primed ROSA^{KIT WT} cells were stimulated by anti-human IgE (5 or 10 µg/mL) or by Cai (1 µM) for 6 hours. Thereafter, cells were centrifuged, and the cell-free supernatants were analyzed for TNF-α content by using a specific EIA kit. For each parameter, data presented are the mean ± SD from 3 independent experiments. *Significantly different from the control (IL-4 + IgE) at P < .05.