Anti human cd3 viogreen

PBMCs were prepared from RA patients before treatment and Cs using Ficoll hypaque (Lymphoprep, Axis-Shield, Oslo, Norway) density gradient centrifugation. In RA patients, the peripheral blood was collected at baseline and was from 4 R-RA and 3 NR-RA patients. PBMCs were stimulated or not with anti-CD3/CD28 beads (Gibco, Waltham, MA, USA) at a bead:PBMC ratio of 1:1. PBMCs were incubated for 72 h with or without 10 ng/mL of recombinant human IL-6 (IL-6) (ImmunoTools, Friesoythe, Germany). The PBMCs were stained with the following monoclonal antibodies: anti-human CD3-Viogreen (Miltenyi Biotec GmBh), CD39-Fitc and CD73-PE (BD Biosciences), and CD8-PECy7 (Biolegend). Samples were acquired using the MACSQuant Analyzer 10 flow cytometer, and the data were analyzed using FlowJo software.

Four hours before stopping the culture, PMA (50 ng/mL) and ionomycin (1 μg/mL) (Sigma-Aldrich) were added. After incubation, the stimulated PBMCs were collected and IFNγ secretion was determined using an IFNγ secretion assay detection kit APC (Miltenyi Biotec GmBh). Additionally, the cells were stained with the following monoclonal antibodies: anti-human CD3-Viogreen (Miltenyi Biotec GmBh), CD8-PECy7, and CD25-APCy7 (Biolegend), CD39-Fitc and CD73-PE (BD Biosciences) as well as violet-fluorescent reactive dye (Thermofisher, Waltham, MA, USA). Samples were acquired with the MACSQuant Analyzer 10 flow cytometer, and the data were analyzed using FlowJo software.