Endotoxin affinity resin

Purified rhIL23R-CHR used in this study was prepared based on our previous report [28 (link)]. The recombinant protein had a purity of 99%. All proteins were treated with Endotoxin affinity Resin (Genscript, USA) to remove the endotoxin before followed assays, the purified proteins were shown to have negligible endotoxin contamination (< 10 EU/mg) by an LAL Chromogenic endotoxin quantization assay(Genscript, USA).

C57BL/6 mice (Yangzhou University Comparative Medical Research Center, Yangzhou, China) spleens were teased through sterilized 70 μm cell strainers (BD Biosciences, San Jose, CA, USA) to obtain single-cell suspensions in IMDM medium (Gibco, Grand Island, NY, USA) containing 10% FBS (Gibco, Grand Island, NY, USA). RBC lysis buffer (BD Pharmingen, San Diego, CA, USA) lysed red blood cells. Anti-CD4 magnetic beads (Miltenyi biotech, Bergisch Gladbach, Germany) was used to purify CD4⁺ T cells. Mixed lymphocyte were stained with APC-adjusted anti-mouse CD4 antibody (BD Biosciences, San Jose, CA, USA) to detect the purity of CD4⁺ T cells. For mouse Th17 cell differentiation, naïve CD4⁺T cells were seeded in 24 well plates at 10⁶/well and stimulated with plate-bound 1 μg/mL anti-mCD3 and 1 μg/mL soluble anti-mCD28 (eBioscience, San Diego, CA, USA) under Th17 polarizing conditions: 1 ng/mL mTGF-β, 10 ng/mL mIL-6, and 10 ng/mL mIL-23 (R&D, Minneapolis, MN, USA) for 72 h. Simultaneous treatment with different concentrations of rhIL-23R-CHR/Fc protein. The endotoxin was removed for all proteins with Endotoxin affinity Resin (Genscript, Piscataway, NJ, USA) before assays.