Apc conjugated cd25

Colon tissue was collected from acute colitis mice and cut into pieces in 1 mg/ml of collagenase IV; collagenase digestion was performed in collagenase solution for 45 minutes at 37 °C. The eluted cells were then passed through a 70-μl cell strainer and collected for 10 minutes at 1400 rpm at room. The cells were incubated in 10% donkey serum and Fc blocked (BD Pharmingen, San Jose, CA) for 20 minutes at 4 °C and then stained with fluorescent-conjugated antibodies. FITC-conjugated CD3, APC-conjugated CD25, PE-conjugated CD4 (OX-38), and APC-conjugated Foxp3 were purchased from BD Pharmingen (San Diego, CA) and used for the FACS analysis. The cells were analyzed with the FACSCaliber cytometer (BD Bioscience, San Diego, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR).

pDCs and mDCs from a HLA-A2.1 + donor were loaded with different concentrations of a melanoma-specific peptide (gp100_280:288) or irrelevant peptide (tyrosinase_369:376) in 96-well round bottom plates (10 × 10³ cells per well). After approximately 2 h, R848, two concentrations of vemurafenib and gp100_280:288-specific T cells (50 × 10³ cells per well) were added. After overnight incubation, CD69 and CD25 expression on the CD3⁺ gp100_280:288-specific T cells was measured by flow cytometry using PE-Cy5 conjugated mouse anti-human CD69, APC-conjugated CD25 and BV421-conjugated CD3 (all BD biosciences).

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.