Percp cy5.5 cd14 clone hcd14

Multiparameter flow cytometric analysis was performed on PBMCs. Briefly, cells were incubated with Fc receptor blocking agent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with monoclonal antibodies for 20 min at 4 °C in a darkened room. CD3 and CD14 immunophenotypic markers were used to define T lymphocytes and monocytes. Each population was also evaluated for CD142 (tissue factor; TF), and PD-L1 expression. The following monoclonal antibodies were used (all from BioLegend, San Diego, CA, USA): FITC-CD3 clone OKT3, PerCP/Cy5.5-CD14 clone HCD14, APC-CD69 clone FN50, PE-CD142 clone NY2, PE/Cy7-HLA-DR clone L243, Brilliant Violet 421-PD-L1 clone 29E.2A3 were used. Matched isotype controls were used for each antibody to establish the gates. Live cells were discriminated by means of LIVE/DEAD Fixable Aqua Dead Cell Stain (Thermo Fisher Scientific, Waltham, MA, USA) and dead cells were excluded from all analyses. All flow cytometric analyses were performed using a BD FACSVerse™ (BD, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).

Immunofluorescence staining was performed on PBMCs. Briefly, cells were incubated with Fc receptor blocking agent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with monoclonal antibodies for 20 min at 4 °C in a darkened room. The following monoclonal antibodies were used (all from BioLegend, San Diego, CA, USA): FITC-CD3 clone OKT3, PerCP/Cy5.5-CD14 clone HCD14, APC-CD69 clone FN50, PE-CD142 clone NY2. After fixation of stained cells using Fix/Perm buffer (Thermo Fisher Scientific, Waltham, MA, USA), the suspension of fixed cells was immobilized onto glass slides by cytospin. Nuclei were counter stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (DOJINDO, Kumamoto, Japan) in water, and whole sections were mounted in ProLong Diamond (Thermo Fisher Scientific, Waltham, MA, USA). Slides were observed with a confocal fluorescence microscope (FV3000, Olympus, Tokyo, Japan).