Bms6001

To stimulate Tregs or effector CD4+ T-cell proliferation, 96 round-bottom plate were coated and incubated overnight at 4° with 10µg/ml anti CD3 antibody. CFSE-stained T-cells were added with or without EXO 10⁸/ml to the plate in complete RPMI 1640 media containing 10µg/ml anti CD28 antibody and incubated at 37° and 5% CO2 for 5days. In some experiments, TGFB1 and IL10 neutralizing antibodies were added. T-cell proliferation was assessed by CFSE dilution using flowcytometry analysis by gating on the CD4+ cells. Regulatory T-cells (Tregs) were identified by gating on double positive cells for FOXP3 (FJK-16s, Thermo fisher Scientific) and CD25 (PC61.5, Thermo fisher Scientific), in CD4+ (GK1.5, Thermo fisher Scientific), population while T-helper 17 induction was identified by measuring IL17 median fluorescent intensity in CD4 population by using anti IL7 Antibody (eBio17B7, Thermofisher scientific), followed by flow cytometry. Supernatants were analysed for IL17 by ELISA (BMS6001, Thermo fisher Scientific). Western blot was used to assess the expression of FOXP3 using antiFOXP3 (FJK-16s, Thermo fisher Scientific).

The concentrations of IL-2, IL-4, IL-10, IL-12, IL-17 and IFN-γ in the culture supernatant of RAW264.7 cells were determined using ELISA kits (cat. nos. BMS601 for IL-2; BMS613 for IL-4; BMS614/2 for IL-10; BMS616 for IL-12; BMS6001 for IL-17; BMS606 for IFN-γ; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. All assays were repeated at least three times.