Fluorospot analysis
Fluorospot assay was performed according to the manufacturer’s protocol. The Fluorospot plate (Mabtech) was coated with 50μg/mL IgE per well at 4°C overnight. First, 1x106 cells from BM and spleen from triple immunized mice were seeded per well. The plate was incubated at 37°C (5% CO2) for 20h. Next, goat anti-mouse IgG biotin primary Ab (SouthernBiotech, Birmingham, AL, USA) diluted in PBS-0.1% BSA (1:1000) was incubated for 2h at RT. Then, streptavidin-550 (Mabtech) diluted in PBS-0.1% BSA (1:200) was added for 1h at RT. Fluorescence enhancer-II (Mabtech) was added for 15 min at RT. The plate was dried at RT overnight. Finally, the plate was read at 550nm using the Fluorospot reader (Mabtech IRIS).