Cd25 alexa 700

Peripheral blood mononuclear cells from HCs and paired PBMCs and SFMCs from RA patients were stained for the presence of T cell co-inhibitory receptors. Non-specific binding was blocked by 50 µg/ml mouse IgG (Jackson), and surface staining was performed using the following antibodies: CD3 V450 (clone: UCHT1, BD), CD4 PE-CF594 (clone: RPA-T4, BD), CD8 BV785 (clone: RPA-T8, BioLegend), CD25 Alexa 700 (clone: BC96, BioLegend), Tim-3 BV711 (clone: F38-2E2, BioLegend), CTLA-4 PerCPCy5.5 (clone: L3D10, BioLegend), Tigit PE-Cy7 (clone: MBSA43, eBioscience), PD-1 APC (clone: MIH4, BD), Live-dead near IR (Thermo Fisher Scientific). Cells were permeabilized by BD Facs lysing solution and BD Facs Perm Solution 2 (both BD bioscience). Intracellular staining was performed using Eomes PE (clone: WD1928 ebioscience). All antibodies were used in the concentration recommended by the manufacturer. Gating was done on lymphocytes, excluding doublets and dead cells. Gates were set using FMOs. CD3⁺ CD4⁺ and CD3⁺ CD8⁺ cells were investigated for their expression of T cell co-inhibitory receptors.

Cells were stained in complete RPMI media or PBS + 2% FBS and 0.1% sodium azide (FACS Buffer) for 30 minutes at 4°C and washed before running on BD LSR-II flow cytometer. Stainings that included antibodies for CCR5 were performed at room temperature. Cell sorting was done using BD FACS Aria (BD Biosciences). Data was analyzed using FlowJo software (Treestar) and gated on live cells based on forward and side scatter properties, as well as fixable viability dye eFluor 450 (eBioscience). The following antibodies were used to stain cells for FACS: HSA-FITC, CD4-APC, CCR5-APC-Cy7 (BD Pharmigen), CD25-Alexa700, CD69-PacificBlue, HLA-DR-APC-Cy7 (Biolegend).