PBMCs were isolated from RA patients using density-gradient centrifugation on Ficoll-Paque. The cell pellets were washed and resuspended with PBS containing 1% FBS. Then, the cells were incubated with FITC-conjugated CD4, PE-Cy5-conjugated CD3, APC-conjugated CD25 and PE-conjugated CD127(all from BD Pharmingen, USA). For intracellular cytokine staining, the cells were pretreated with Cell Activation Cocktail (Thermo) for 6h. Then, cells were washed and stained with PE-conjugated CD4 and AF750-conjugated CD3 antibodies (all from BD Pharmingen, USA). After fixed and permeabilized with Transcription Factor Buffer Set (BD Pharmingen, USA), the cells were stained with AF700-conjugated IL-17A (Biolegend, USA), FITC-conjugated IFN-γ and PE-Cy7 conjugated IL-4 antibodies (all from BD Pharmingen, USA). For Foxp3 staining, cells were fixed and permeabilized, and then stained with BV421 conjugated Foxp3 antibody (Biolegend, USA). Multiparameter flow cytometry (DxFLEX, Beckmancoulter) and FlowJo software (Tree Star) were adopted for the data analysis of stained cells.
Cytometric bead array (CBA) was used to measure IL-6, IFN-γ, IL-4, IL-17A and IL-10 levels in serum from RA patients or health controls. The measurement was performed using Aimplex Human Th1/Th2/Th17 14-plex cytokine kit (Quanto Bio, China) according to the manufacture’s instruction.
Cytometric bead array (CBA) was used to measure IL-6, IFN-γ, IL-4, IL-17A and IL-10 levels in serum from RA patients or health controls. The measurement was performed using Aimplex Human Th1/Th2/Th17 14-plex cytokine kit (Quanto Bio, China) according to the manufacture’s instruction.