In vitro transcytosis was performed as previously described with modifications (16 (link)). Tracheal epithelial cells from WT or CD23 KO mice were grown onto 0.4-µm-pore size Transwell inserts (BD Biosciences) to form a monolayer exhibiting transepithelial electrical resistances (1500 Ω/cm2). TER was measured using a tissue-resistance measurement equipped with planar electrodes (World Precision Instruments, Sarasota, FL). Monolayers were equilibrated in serum-free DMEM. Mouse IgE was added to either the apical (50 µg/250 µl) or the basolateral (50 µg/500 µl) side. They were incubated at 37°C for 2 h. For negative control, monolayers were also incubated at 4°C for 1 h before transcytosis. The media from the opposite chambers were collected and IgE was measured by ELISA.
In-vivo transcytosis was performed with a set of WT and CD23 KO mice. Mouse OVA specific IgE (50 µg/40µl) in PBS was given either intranasally (i.n.) or intraperitoneally (i.p.). 4, 8 or 24 h later, serum or bronchoalveolar lavage (BAL) fluids were sampled, respectively. Immune complexes (ICs) formed with mouse OVA specific IgE (20 µg) and chicken OVA (10 µg/40 µl) in PBS for 30 min at room temperature. Mice were i.n. inoculated with ICs or chicken OVA antigen alone (10 µg/40 µl) in PBS; 8 h later, sera were sampled. The concentration of OVA-specific IgE and OVA were determined by ELISA.
In-vivo transcytosis was performed with a set of WT and CD23 KO mice. Mouse OVA specific IgE (50 µg/40µl) in PBS was given either intranasally (i.n.) or intraperitoneally (i.p.). 4, 8 or 24 h later, serum or bronchoalveolar lavage (BAL) fluids were sampled, respectively. Immune complexes (ICs) formed with mouse OVA specific IgE (20 µg) and chicken OVA (10 µg/40 µl) in PBS for 30 min at room temperature. Mice were i.n. inoculated with ICs or chicken OVA antigen alone (10 µg/40 µl) in PBS; 8 h later, sera were sampled. The concentration of OVA-specific IgE and OVA were determined by ELISA.