Anti cd4 percp clone sk3

Staining with anti-HLA-A2-PE (clone BB7.2, BD Biosciences, cat. no. 558570), anti-HLA-A2-FITC (clone BB7.2, BD Biosciences, cat. no. 551285), anti-CD4-PerCP (clone SK3, BD Biosciences, cat. no. 345770), and/or anti-CD25-PE (clone 4E3, Miltenyi Biotec; cat. no. 130-091-024) was performed in the dark with antibody dilutions in FACS buffer (PBS/0.5% HSA) for 15 min at 20°C or 30 min at 4°C. Cells were washed once with PBS, resuspended, and measured in FACS buffer. For intracellular FOXP3 staining, cells were first stained with surface antibodies as described earlier, followed by fixable viability dye-eFlour780 (ebioscience, cat. no. 65-0865-14) staining enabling gating on live cells. Subsequently, fixation, permeabilization, and intracellular staining was performed with the Foxp3 Staining Buffer Set (ebioscience, cat. no. 00-5523-00) using anti-FOXP3-APC (clone 236A/E7, ebioscience, cat. no. 17-4777-42) or equally concentrated mIgG1κ APC isotype control (clone P3.6.2.8.1, ebioscience, cat. no. 17-4714-42) antibodies. Acquisition was performed on a CyAn ADP 9 Color Analyzer (Beckman Coulter), and parameter compensation was performed automatically with the CyAn software (Summit) tool using single stained samples containing positive cells. Flow cytometry data were analyzed using the FlowJo software (Tree Star).

The phenotype of CD4⁺ T cells was determined based on CD25 and Foxp3 markers. Surface antigens CD4 and CD25 were stained with anti-CD4-PerCP (clone SK 3, BD Biosciences, USA) and anti-CD25-FITC (clone M-251; BD Biosciences) monoclonal antibodies in the dark for 20 min at 4 °C. Following staining, the cells were washed twice with PBS and stained intracellularly with Foxp3-PE Staining Kit (clone PCH101; eBioscience, USA) according to manufacturer’s protocol. Acquisition was performed on BD Accuri C6 (BD Biosciences). FACS data were analyzed using FlowJo.

Staining with anti‐HLA‐A2‐PE (clone BB7.2, BD Biosciences), anti‐HLA‐A2‐FITC (clone BB7.2, BD Biosciences), anti‐CD4‐PErCP (clone SK3, BD Biosciences), anti‐CD4‐PE (clone #11830, R&D systems), anti‐CD25‐PE (clone 4E3, Miltenyi Biotec), and/or anti‐CD3‐PE‐Vio770 (clone BW264/56, Miltenyi Biotec) was performed in the dark with Ab diluted in FACS buffer (PBS with 0.5% HSA) for 15 min at 20°C or 30 min at 4°C. Cells were washed once with PBS, resuspended, and measured in FACS buffer. Where noted, following surface staining, viability staining was performed with Fixable Viability Dye eFluor 780 (eBioscience) according to the manufacturer's instruction (without subsequent fixation). Live cells were gated via fsc/ssc and, where applicable, on viability dye‐negative cells. Backgating of viability dye positive and negative populations confirmed that dead cells could be completely gated out by fsc/ssc with the purified CD4 T cells used. Acquisition was performed on a CyAn ADP 9 Color Analyzer (Beckman Coulter) or BD FACSVerse (BD Biosciences), and automatic parameter compensation was performed automatically with the CyAn software (Summit) tool or with FlowJo utilizing single stained control samples. Flow cytometry data were analyzed using FlowJo software (Tree Star) version 10.4.1.