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).