Anti igg1 apc

CSR to IgG1 was performed as previously described in Refs 4, 8, 24. Briefly, splenic B cells were isolated by negative selection using magnetic immunolabelling with an EasySep™ Mouse B Cell Isolation Kit (Stemcell, Cambridge, UK, #19854), stimulated with IL‐4 and lipopolysaccharide (LPS), and analyzed by flow cytometry at day 4. Antibody used for IgG1 detection was anti‐IgG1‐APC (BD Biosciences, Franklin Lakes, NJ, USA, #550874). Primary murine tail fibroblasts were generated and cultured as previously described 4, 5, 7. T‐FISH was performed as previously described 4, 5, 7. Statistical analyses were performed using graphpad prism 7.03 (La Jolla, CA, USA), one‐way ANOVA.

Freshly isolated bulk splenocytes from SW_HEL mice containing 10⁴ HEL⁺ B cells were transferred into C57BL/6 mice by intravenous injection, as previously described [50] (link). Sheep red blood cells (SRBC) were obtained from Miguel Fevereiro, Laboratório Nacional de Investigação Veterinária, Lisbon. Recombinant HEL was covalently conjugated to SRBC with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (Sigma-Aldrich) as described [25] (link). Conjugation was confirmed by FACS by staining mock or HEL-conjugated SRBC with the HyHEL10 (an anti-HEL IgG₁[21] (link)) followed by an anti-IgG₁ APC (BD Biosciences). HEL-A647 was used to identify transferred HEL⁺ B cells.

Antibodies were purchased as followed: Anti-CD4-V450, anti-CD8α-APCcy7, anti-B220-V450, anti-CD138-Pe-cy7, anti-CD86-Pe-cy5, anti-FAS-PE, anti-GL-7-APC, anti-CD40-APC, anti-MHCII-FITC, anti-IL-4-PE, anti-IL-5-PE, anti-IgM-Pe-cy7, anti-IgG1-APC, anti-IFN-r-Percp5.5, anti-TNF-α-PE, and anti-IL-17-Pe-cy7 were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-PDL-1-biotin (eBioscience, San Diego, CA), anti-XBP-1s (Cell Signaling Technology, Danvers, MA), and anti-Rabbit IgG-FITC (Thermo Fisher Scientific, Waltham, MA) antibodies were purchased from commercial sources. Recombinant mouse IL-4 (PeproTech, Rocky Hill, NJ) and LPS (Sigma-Aldrich, St. Louis, MO) were purchased from the commercial companies. Goat F(ab’)2 Anti-Mouse IgM (1022-01, SouthernBiotech, Birmingham, AL) and anti-mouse CD40 (BE0016-2, BioXCell, Lebanon, NH) were commercially purchased from companies.