T cell phenotype, proliferation, and cytokine secretion.
TransAct
PROLIFERATION
Thawed PBMCs were stained with Fixable Viability Stain 520 (FVS520, BD Biosciences) and then incubated with the fluorochrome-conjugated monoclonal antibodies (see Proliferation of T cells was evaluated after labeling PBMCs with 10 μM CellTrace CFSE (Thermo Fisher Scientific) and then incubating the cells for 6 days in TexMACS medium with and without T cells TransAct (Miltenyi Biotec) at 37°C and 5% COLevels of IL-10, IFN-γ, and IL-17A (as pg/mL cells) were assessed in the supernatant of proliferating T cells by ELISA (Thermo Fisher Scientific).All samples (for both B cell and T cell analyses) were acquired by multicolor flow cytometry (FACS Fortessa X-20, BD Biosciences) and analyzed by FlowJo software, version 10.7.1 (BD Biosciences); conjugated antibodies and cells viability markers are detailed in 