The cell types were assessed phenotypically by flow cytometry and morphologically after H and E-staining. For analysis, 106 cells suspended in 50 μL of cold PBS containing 2% FCS were stained

with a fluorescein-labeled rat anti-mouse Ab (1 μg) in the presence of 1.5 μL of rat whole serum and 1 μg of purified rat anti-mouse FcγRIII/II mAb (PharMingen) at 4°C for 20 min, washed, and analyzed by use of FACS. For sorting, the fluorescein-labeled or unlabeled cells were isolated by FACS in forward scattering/side scattering and FITC/PE modes, as described previously (16). Cell numbers were determined by counting the cells in Turk’s solution with a hemocytometer. Cell viability was assessed by the trypan blue exclusion method. Cells in the lymphocyte-, macrophage- or granulocyte-rich

fractions or various combinations of cells in the Afatinib lymphocyte-rich fraction with specific antigen + or − cells were cultured for 6 days without cedar pollen in a 24- or 48-well plate containing RPMI 1640 medium containing Metformin mouse 10% FCS. The culture media were stored in microtubes at − 20°C prior to use. The wells of enzyme immunoassay/radioimmunoassay (Costar #2592; Corning, NY, USA) were coated with 100 μL of rat anti-mouse IgE or anti-IgG Ab (each 2 μg/mL) at 4°C overnight. After three washes with PBS/Tween (PBS + 0.5% Tween 20), the wells were filled with 400 μL of 1% BSA/PBS and then incubated for 2 hr at room temperature to block unsaturated protein binding sites. The plates were next washed three times with PBS/Tween; and 100 μL of appropriately diluted serum samples in 1% BSA/PBS added to each of triplicate wells, after which the plates were incubated for 2 hr at room temperature. After three more washes with PBS/Tween, 100 μL of HRP-labeled goat anti-mouse Cyclooxygenase (COX) IgE or IgG Ab in 1% BSA/PBS was dispensed into each well, and the preparation allowed

to react for 1 hr at room temperature. The wells were then washed thoroughly with PBS/Tween. Next, the antigen-Ab complex was incubated with 100 μL of tetramethylbenzidine (Sigma-Aldrich) for 30 min at room temperature. The reaction was then stopped by the addition of 100 μL of 1M H2SO4. Thereafter the OD450nm of the solution in each well was read by a microplate reader SH-1000 (Corona Electric, Hitachinaka, Japan). For preparation of standard curves for serum Igs, the concentrations used in this study were as follow: 0.0039 μg/mL, 0.0078 μg/mL, 0.0156 μg/mL, 0.0313 μg/mL, 0.0625, and 0.125 μg/mL for IgG and 0.00156 μg/mL, 0.00313 μg/mL, 0.00625 μg/mL, 0.0125 μg/mL, and 0.025 μg/mL for IgE. To measure the Ig concentrations, serum samples were diluted 20-, 50-, and 100-fold for IgE and 50000-, 10,0000-, and 20,0000-fold for IgG.