Cells were rapid desensitized as CP-673451 per Table 1. After desensitization (nearly 2 h) cells were maintained for 10 min, 2 hours, or 4 hours at 37°C. After each time period, 1 ng of DNP-HSA or 25 μL of calcium ionophore A23187 (Sigma-Aldrich) 10 μM was added. Non-desensitized cells were kept at 37°C and challenged with 1 ng of DNP-HSA or 1 ng HSA at the same time points as for desensitized cells. The total time for all cells at 37°C, since rapid desensitization protocol lasts nearly 2 h, was 6 h. Cell viability

was assessed by trypan blue dye exclusion. After desensitization or challenge, cells were collected and washed with cold PBS. Pellets were lysed in RIPA buffer supplemented with protease and phosphatase

inhibitor cocktails (Roche). Total protein lysates were subjected to SDS-PAGE on a 4–12% polyacrylamide gel and transferred to a nitrocellulose membrane (both from Invitrogen). Membranes were blotted with anti-Phospho-STAT6 (phosphotyrosine 641) and anti-STAT6 from Sigma-Aldrich or with anti-Phospho-p38MAP kinase and anti-p38αMAP kinase from Cell Signaling. Signal detection was performed with SuperSignal West Pico Chemiluminescent Substrate (Pierce). After desensitization or challenge, cells were placed at 4°C, then washed and resuspended in PBS containing 0.5% BSA and 0.05% sodium azide at 4°C and incubated with anti-FcγRI/II mAb (eBioscience) for 20 min on ice to block Fcγ receptors. Cells were then incubated with selleck chemicals llc 5 μg/mL FITC rat anti-mouse IgE (BD Biosciences) or 2 μg/mL PE Armenian hamster anti-mouse FcεRIα HSP90 (eBioscience) or with the recommended isotype controls. Cells were analyzed on a BD Biosciences FACSCanto flow cytometer, using FACSDiva acquisition software and FlowJo analysis software.

Antigens used were Alexa Fluor 488-conjugated OVA (Molecular Probes) and DyLight Fluor 649-conjugated DNP, labeled with DyLight 649 NHS Ester (Thermo Scientific). Due to detection limitations, OVA activation dose was 50 ng, DNP activation dose was 5 ng and the rapid OVA desensitization protocol was consequently adjusted based on the volumes used in the protocol in Table 1 but at higher concentrations. After desensitization or challenge, cells were washed and resuspended in cold PBS. Cells were transferred onto poly-L-lysine-coated round cover slips for 20 min at 4°C and then fixed with 4% paraformaldehyde in PBS for 10 min at 4°C. After three washes with PBS, cells were incubated with cholera toxin subunit B-Alexa Fluor 555 conjugate (Molecular Probes) 1:500 in PBS for 10 min at 4°C, washed three times with PBS and mounted using an aqueous mounting medium (15% wt/v polyvinyl alcohol, 33% v/v glycerol, 0.1% azide). Images were collected sequentially using a 63× plan Apo NA 1.4 objective on Leica SP5X laser scanning confocal system attached to an inverted Leica DMI6000 microscope.