Anti human cd3 pe

PBMCs were separated by Ficoll^® Paque in Leucosep tubes and washed twice in PBS containing 0.5% bovine serum albumin (BSA) (all from Sigma-Aldrich, St. Louis, MO, USA). Dimethyl sulfoxide cryo-conserved PBMCs were thawed in RPMI 1640 medium supplemented with PBS and fetal bovine serum (all from Sigma-Aldrich, St. Louis, MO, USA), washed, and resuspended in PBS/BSA. Cell suspensions were stained in PBS containing specific antibodies and 2% Beriglobin for 15 min at 4 °C, followed by washing in PBS and fixation in 4% paraformaldehyde for 10 min at 37 °C. Intracellular staining of cytokines was performed after fixation in the presence of 0.5% saponin (all from Sigma-Aldrich, St. Louis, MO, USA). The antibodies used were anti-human CD3 PE, IL-6 FITC (both from Becton, Dickinson and Company, Franklin Lakes, NJ, USA), HLA-DR Per-CP, PAR1-Alexa488 (both from R&D Systems, Minneapolis, MN, USA), and TNF-α APC Vio-770 (Miltenyi Biotec, Bergisch Gladbach, Germany). The percentage of FIIa-activated PAR1-positive (the antibody detects fragments of activated PAR1-cleaved-Ser42 protein) (Sigma-Aldrich, St. Louis, MO, USA) (visualized with goat anti-rabbit IgG Alexa 405; Thermo Fisher Scientific, Waltham, MA, USA) circulating PBMCs was measured by using flow cytometry with FACSCaliburTM and CellQuest software 5.1 (both from Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

Peripheral blood mononuclear cells (PBMCs) were prepared as previously described [37 (link)]. Thawed PBMC were fixed for 10 minutes with 4% formalin followed by staining with anti-human CD3 PE (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and PAR1-Alexa488 (R&D Systems, Minneapolis, MN, USA). The percentage of FIIa-activated PAR1-positive (the antibody detects fragments of activated PAR1-cleaved-Ser⁴² protein) (Sigma-Aldrich, St. Louis, MO, USA) circulating CD8⁺ T cells (anti-human CD8 PerCP from Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was measured using flow cytometry with a FACSCalibur^TM and CellQuest software (both Becton, Dickinson and Company, Franklin Lakes, NJ, USA) [18 (link),19 (link),38 (link)]. The intracellular staining of cytokines after PBMC stimulation was performed as previously described [37 (link),39 (link)].

Antibodies for surface markers staining of DCs and T cells were obtained from BD Biosciences, New York, USA (anti-human CD3-PE, CD3-FITC, CD8-PerCP, CD8-APC, CD56-PE, NKG2D-APC, CD4-FITC, CD4-PerCP, CD107a-FITC, CD25-APC, CD45RO-FITC, CD27-PerCPCY5.5, CD57-APC, CCR7-PE, CD14-APC, CD80-PE, CD83-APC, CD86-FITC, HLA-DR-FITC). Antibodies for intracellular proteins staining were also obtained from BD Biosciences (anti-human IFNγ-APC, TNFα-PECY7, granzyme B-FITC, FoxP3-PE). The intracellular staining was performed by fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences). The experiments were performed by using FACS CantoII (BD Biosciences) flow cytometer, and data were analyzed by using the Flowjo software.