Legendplex mouse immunoglobulin isotyping panel

Total IgG1 (tIgG1) levels were analyzed using the LEGENDplex™ Mouse Immunoglobulin Isotyping Panel (BioLegend), according to the manufacturer’s instructions.
For the detection of OVA-specific IgG1 (OVA-sIgG1), plates were coated with 1 µg/mL ovalbumin grade V (Merck). OVA-sIgG1 was detected with a biotinylated anti-mouse IgG1 antibody (BD Biosciences).
Quantification of total IgE (tIgE) was performed with the BD Mouse IgE ELISA set (BD Biosciences), following the manufacturer’s instructions.
OVA-specific IgE (OVA-sIgE) was analyzed using the LEGEND MAX™ Mouse OVA Specific IgE ELISA Kit (BioLegend), according to the manufacturer’s instructions.

To assay for total plasma IgE, 96-well Nunc MaxiSorp plates (44-2404-21; ThermoFisher) were coated overnight at 4°C with 2 µg/ml rat anti-mouse IgE (553413; BD Biosciences) diluted in 0.1 M pH 9.6 carbonate-bicarbonate buffer (C3041; Sigma-Aldrich). Plates were washed in 1× PBS with 0.05% Tween 20, blocked in 1× PBS with 1% BSA at room temperature for 1 h, and then incubated for 2 h at room temperature with plasma or serially diluted mouse IgE κ isotype control (557080; BD Pharmingen), all diluted in 1× PBS with 1% BSA. For detection, the washed plates were incubated with 0.5 µg/ml biotinylated rat anti-mouse IgE (553419; BD Biosciences) for 1 h at room temperature, washed, and incubated for 30 min at room temperature with streptavidin–HRP (1:1,000, 554066; Thermo Fisher Scientific), all diluted in 1× PBS with 1% BSA. The washed wells were developed with TMB substrate solution (555214; BD Biosciences), quenched with 3 M H₂SO₄ (258105; Sigma-Aldrich), and detected at 450 nm by spectrophotometer for optical density and back-calculated concentration (SpectraMax M5). Other antibody isotypes were assayed with the LEGENDplex Mouse Immunoglobulin Isotyping Panel by flow cytometry (740493; BioLegend). Cort levels from plasma collected at Zeitgeber time 4 (ZT4) or from cell lysates of ileal epithelium were assayed by the DetectX Corticosterone Enzyme Immunoassay Kit (K014-H1; Arbor).

Human serum concentrations of CXCL9, CXCL10, and CXCL11 were measured using the LEGENDplex Human Proinflammatory Chemokine Panel (BioLegend) according to the manufacturer’s instructions. Human serum concentrations of other cytokines were measured using the 42-Plex Human Cytokine/Chemokine Array (Eve Technologies). Mouse serum antibody concentrations were measured using the LEGENDplex Mouse Immunoglobulin Isotyping Panel (BioLegend). Mouse serum cytokine and chemokine concentrations were measured using the LEGENDplex Mouse Inflammation Panel (BioLegend). Cytokine concentrations from cell culture supernatant were measured with the Mouse IL-12 (p70) ELISA MAX Deluxe, Human IL-12 (p70) ELISA MAX Deluxe, and LEGEND MAX Human IL-23 ELISA Kit (all from BioLegend).