Mouse isotyping panel 1 assay kit

Plasma IgA, IgG1, IgG2a, IgG2b and IgM levels were assessed with the use of Mouse Isotyping Panel 1 Assay kit from Mesoscale according to the company’s instructions (MSD Multi-spot Assays, Mesoscale Discovery, Rockville, Maryland). Plasma IgM antibodies targeting PC were detected with an ELISA kit from Athera Biotechnologies (Athera Biotechnologies AB, Solna, Sweden) according to the company’s instructions, with the exception of using peroxidase-conjugated anti-mouse IgM (Jackson ImmunoResearch, Novakemi, Handen, Sweden).

Four-week-old F1 mice (5–8/treatment group) were selected for early-life immune assessment. Mice were anaesthetized with isoflurane and blood was obtained by cardiac puncture after confirmed absence of reflex. Following cardiac puncture, mice were sacrificed by cervical dislocation and spleen, jejunum, and colon sections were immediately removed for flow cytometry or cytokine measurements as described below. Blood was allowed to clot on ice, spun at 3000 × g for 10 min, and supernatant (serum) was used to determine immunoglobulin levels. IgE titer was determined in IgE BD OptEIA ELISA (BD Biosciences, San Diego, CA, USA). IgA, IgG1, IgG2a, IgG2b, IgG3, and IgM titers were determined in Mouse Isotyping Panel 1 Assay Kit (Meso Scale Discovery). Jejunum and colon fragments were flash-frozen for cytokine measurements in U-Plex T Cell Combo Assay Kit (Meso Scale Discovery).

Plasma IgA, IgG1, IgG2a (not expressed in C57BL/6 mice), IgG2b, IgG3 and IgM levels were assessed with the use of Mouse Isotyping Panel 1 Assay kit from Mesoscale according to the company’s instructions (MSD Multi-spot Assays, Mesoscale Discovery, Maryland, USA). Plasma IgE levels were determined with the use of mouse IgE ELISA Kit from Bethyl Laboratories (Bethyl Laboratories Inc, Montgomery, Texas, USA) while IgM antibodies targeting phosphorylcholine were assessed with an ELISA kit from Athera Biotechnologies (Athera Biotechnologies AB, Solna, Sweden) according to the company’s instructions, with the exception of the use of peroxidase-conjugated anti mouse IgM (Jackson ImmunoResearch).