Vegfr2 pe

Circulating EPCs were quantified using the method devised by Duda et al. [42 (link)], with a few modifications described in our previous study [43 (link)]. Flow cytometry was used for whole blood analysis without enrichment procedures to avoid manipulation artifacts. The EPCs were characterized as CD31⁺/vascular endothelial growth factor-2 (VEGFR-2)⁺/CD45^dim/CD133⁺. The chromophores conjugated with specific antibodies used in this study included CD31-FITC (BD Pharmingen, San Diego, CA), VEGFR2-PE (BD Pharmingen), CD45-PerCP (BD Pharmingen), and CD133-PE (Miltenyi Biotec, Auburn, CA). During the analysis of flow cytometry data, the mononuclear cell population was gated to avoid red blood cell, platelet, cell debris, and neutrophil contamination; 100,000 events in the gated population were collected using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). The acquisition data were collected and analyzed using CellQuest Software (BD Biosciences). Representative data for identification and quantification of EPCs by flow cytometric analysis is shown in Figure 4.

HUVEC were incubated with TNFα (25ng/ml) and two ratios of MP (1:50,000, 1:100,000 HUVEC : MP) during 24h. Then, the cells were trypsinized and washed with FACS Flow (BD Biosciences, San Jose, CA). The immunophenotypic characterization of the activation state of endothelial cells was done by incubating HUVEC with mouse-anti-human monoclonal antibodies against CD54-APC, CD106-BV421, CD62e-PE, CD31-FITC, VEGFR2-PE, CD105-FITC and TIE2-Alex647 (all BD Biosciences). All the antibodies were incubated with the cells for 30 min, at room temperature in the absence of light. After two washes with FACS Flow, flow cytometric analysis was performed using FACSCANTO-II with FACSDIVA Software (BD Biosciences).

Single-cell suspensions were generated by digestion of confluent cells. A total of 10⁶ cells were incubated with antibodies or an isotype control antibody at 4°C for 30 min in the dark. Samples were washed twice with PBS and analyzed with an FACS Calibur flow cytometer (Becton Dickinson). Specific antibodies used in these analyses were CD34-PE, CD29-PE, CD90-PE, CD45-FITC, CD73-FITC, CD105-FITC, CD14-PE, CD19-PE, CD4-PE, CD8-APC (Biolegend, USA), VEGFR-2-PE, CD144-PE (BD, USA), vWF-FITC (Abcam, USA), CD31-FITC (eBioscience, USA), MHC I-PE, MHC II-PE, CD40-PE, CD80-PE, CD86-PE (Miltenyi Biotec) and isotype control IgG-PE (from Miltenyi Biotec or Biolegend, USA), IgG-FITC (from ebioscience or Biolegend, USA), Flow cytometric data were analyzed using BD CELLQuest software.