Anti mouse il 10 antibody

Anti-mouse TLR2 antibody at a concentration of 1 mg/mL (BioLegend) and specific chemical inhibitors for ERK1/2 MAPK (PD98059) and p38 MAPK (SB203580) (Selleck Chemicals LLC, Houston, TX, USA) at a concentration of 10 μM in assays were precultured with RAW264.7 cells for 2 h before stimulation with bifidobacterial strains. Anti-mouse IL-10 antibody (BioLegend) was added to the coculture of bifidobacterial strains and splenocytes.

Thymus tissue samples were prepared (paraffin-embedded and frozen sample), and 5-μm sections were stained, as previously reported (29 (link)), with anti-mouse IL-10 antibody (Biolegend, San Diego, USA). Imaging was done by ZEISS Primo Star microscope, Zeiss (Zena, Germany), and laser capture micro-dissection was done by ZIESS PALM/APOSTOME (Zena, Germany) laser capture microscope.

For B-T cell co-culture experiments B cells served as APCs and were stimulated for 24 h with commensals as described previously (“-primed B cells”). Prior to co-cultivation with naïve CFSE-labeled OT-II CD4⁺ T cells, B cells were incubated with 10 μg/mL Ova-Peptide (ISQAVHAAHAEINEAGR, EMC) for 2 h at 37°C. B cells were washed and supernatant was exchanged with fresh media (“-pulsed B cells”). Primed and pulsed B cells and naïve T cells were co-cultured at different ratios for 72 h at 37°C and 100 ng/mL purified anti-mouse IL-10 antibody (Clone: JES5-2A5, BioLegend) was added to certain samples.
For CD11c⁺ dendritic cells maturation assay, naïve B cells and differentiated immature BMDCs were simultaneously stimulated with bacteria at MOI 1 and co-cultured at a ratio of 5:1 (B cells/BMDCs). To show the effect of indirect cell-cell interaction, naïve B cells and BMDCs were additionally co-cultured in Transwells (pore-size 0.4 μm) and stimulated with bacteria at MOI 1. To mimic the influence of soluble IL-10 produced by B cells on the maturation of BMDCs, 10 μg/mL recombinant mouse IL-10 (BioLegend) were added to BMDC mono-cultures.