Duoset elisa

The IL-1β, IL-5, IL-6, and IL-12 levels were measured using Ready-SET-Go! ELISA (eBioscience, San Diego, CA), RANTES, and IFN-γ levels were measured using DuoSet! ELISA (R & D Systems, Abingdon, UK) and IgE levels were measured using an ELISA kit (BD Pharmingen) according to the manufacturers' protocols. Der p-specific antibodies were measured in a similar manner as IgE levels using 2 μg/ml Der p antigen instead of the capture antibody and biotinylated rat anti-mouse IgG1 or IgG2a/2b mAb (2 μg/ml; BD Pharmingen). After the completion of the color reaction, the result was scanned and recorded by using an ELISA recorder at OD₄₅₀.

B cells were isolated from spleens of individual mice using the EasySep mouse B cell isolation kit (STEMCELL Technologies). B cell purity was more than 90% as determined by FACS using CD19 and CD45R/B220 (Supplemental Table 2). Isolated B cells (200,000 cells/well) were stimulated with TLR agonists as indicated in Supplemental Table 3. IL-6 was quantified by DuoSet ELISA (eBioscience) using a SpectraMax plate reader with SoftmaxPro V5 software (Molecular Devices).
For cell signaling studies, pooled B cells (2 × 10⁶ cells) were stimulated with TLR agonists at times indicated in the figures prior to lysis in RIPA buffer (MilliporeSigma) containing protease and phosphatase inhibitors (MilliporeSigma). Protein expression of IRAK1, IRAK4, NF-κB p65, p-p38, and total p38 was analyzed by automated capillary-based immunoassay system (WES) using standard protocols (ProteinSimple). In brief, each lysate was diluted with 5× Fluorescent Master Mix (ProteinSimple) and incubated with each primary antibody together with appropriate loading control as indicated in Supplemental Table 2. Samples were loaded onto a 12–230 kDa WES Separation Module 25 capillary cartridge and quantification of each protein was determined using the Compass software v6.0.0 (ProteinSimple).