Anti human cd3 bv421

Frequencies of B cells, CD4⁺ T cells, and CD8⁺ T cells in human PBMC were determined by flow cytometry, as previously described [8 (link)]. Briefly, human PBMC with or without T or B cell depletion were stained with anti-human CD3-BV421 (BD Biosciences, clone UCHT1), anti-human CD4-BV650 (BD Biosciences, clone 2RPA-T4), anti-human CD8-APC (BD Biosciences, clone SK1), anti-human CD20-Percp/cy5.5 (BD Biosciences, clone 2H7), and anti-human CD45-FITC (BD Biosciences, clone 2D1) and measured by using LSR II flow cytometer (BD, USA). The data were analyzed using the FACS Express software (De Novo Software, USA, version 5).

Whole blood or PBMC were stained with antibody mix comprising anti-human CD3-Bv421, CD4-PE, CD8-APC, CD14-APCH7, CD19-Bv480, CD20-PECy7, and CD45-FITC (clones UCHT1, SK3, SK1, MphiP9, SJ25C1, L27, 2D1, respectively) and 7-AAD (all from BD Biosciences). For whole blood, BD Trucount™ tubes were used according to the manufacturer’s instructions (Figure S1). Briefly, antibody mix was added followed by 50 μl whole blood by reverse pipetting. Tubes were vortexed and incubated for 15 min at room temperature in the dark. Erythrocytes were lysed by incubating with 450 μl 1xPharm Lyse™ (BD Biosciences), for 15min before analysing samples on a BD FACS Verse.

Peripheral blood mononuclear cells were thawed and rested as described above. 10⁶ cells were placed per tube and washed with phosphate buffered saline (PBS) (#L0615-500, Biowest), 400 g for 5 min at 4°C before staining with Fixable Viability Dye eFlouro 506 (#65-0866, eBioscience) for 20 min at RT. Cells were blocked for unspecific binding in PBS with 10% human serum (#H4522, Sigma-Aldrich) for 15 min on ice. Then, they were washed with PBS and incubated with combinations of extracellular anti-human CD3-BV421 (#563798, BD Bioscience), anti-human CD4-FITC (#11-0047-42, eBioscience), or CD8-PE-Cy7 (#344712, Nordic BioSite) for 30 min at 4°C. The cells were washed in PBS with 0.5% human serum before fixation and stained as previously described (16 (link)). Briefly, human P2Y₁₁ receptor staining was performed on unpermeabilized cells with 0.1-µg Lightning Link (#705-0030, Innova Biosciences, UK) APC-conjugated 1:100 rabbit anti-human P2Y₁₁ receptor antibody (#ab140878, Abcam) against the extracellular part for 30 min. We have previously tested this antibody and found it specific for the detection of human P2Y₁₁ over P2Y₁ and P2Y₂ in transfected cells by flow cytometry (16 (link)). Flow cytometry was done on FACSverse (BD Biosciences, USA), and the FACSuite software (BD Biosciences, USA) was used for data analysis.