Anti human cd3 percp

For cell surface and intracellular staining, standard protocols were followed using monoclonal antibodies. Cells and antibodies were co-incubated on ice in the dark for 30 minutes. For intracellular staining of cytokines, cells were permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA, USA) for 30 minutes on ice, followed by an additional 30-minute incubation with antibodies in the dark at 4°C. The following antibodies were used: Anti-HLA-DR-APC (BD, cat. no.: 339194), anti-human CD54-APC (BD, cat. no.: 561899), anti-human CD86-FITC (BD, cat. no.: 560958), anti-human CD3-Percp (BD, cat. no.: 552851), anti-Human CD4-PE (BD, cat. no.: 557344), anti-Human CD8-FITC (BD, cat. no.: 555366), anti-human Ki-67-APC (BioLegend, cat. no.: 350514), and anti-IFN-gamma-APC (BD, cat. no.: IC285A-100). Imaging flow cytometry was performed using the ImageStreamX MarkII quantitative imaging flow cytometer, while conventional flow cytometry was performed using the BD FACS Calibur flow cytometer.

Phenotypic analyses of T cells and B cells were performed with anti-human monoclonal antibodies (mAbs): anti-human CD3-PerCP, CD4-FITC, CD19-PerCP and CD21-APC were from BD Biosciences (San Jose, CA, USA). CXCR5-APC, ICOS-PE, PD-1-PE, IFN-γ-PE, IL-4-PE, IL-17-PE, IL-21-PE, IL-22-PE, CD27-FITC, CD86-PE, CD95-PE, CD25-APC, and Foxp3-PE antibodies were obtained from eBiosciences (San Diego, CA, USA). Cells were analyzed by flow cytometry (BD FACSCalibur, San Jose, CA) and data was analyzed by FlowJo software (Tree Star, San Carlos, CA).

Cells were stained with relevant antibodies on ice for 30 minutes in PBS buffer containing 2% FCS and 0.1% sodium azide. Before staining for surface markers, cells were incubated with Fc Blocker (CD16/CD32) for 30 minutes to minimize non-specific staining. Cells were washed twice before being analysed by BD FACS ARIA II flow cytometer. Live cells were gated based on forward and side scatter profiles and based on exclusion with live/dead aqua marker (Invitrogen). The following antibodies were used for staining: anti-human CD3 PerCP, CD4 APC or CD4 PE-Texas red, CD25 APC-Cy7, CD127 PE-Cy7, GARP PE (All from BD Biosciences) and anti-human Latency associated peptide (LAP)-TGF-β1 Alexa Fluor 488 (R and D systems). Recombinant human LAP (rLAP) that associates with TGF-β1 was purchased from R and D systems. Analysis was performed using FlowJo software (Tree Star).
The Tr1 and iTreg cells were sorted on the BD FACS ARIAII Flow Cytometer aseptically for further experiments. 2.5 million PBMCs from each well of tissue culture plate were sorted in approximately 5 minutes. Sorted cells were collected in 12×75 mm polypropylene tubes pre-coated with human AB serum and containing complete RPMI-1640 with 10% human AB serum.