Cd31 fitc

Cell surface antigens on cells were evaluated with FACS. The cells were dissociated with 0.05% trypsin/EDTA (Highclone), washed with PBS, fixed with 4% paraformaldehyde, permeabilized with Triton X‐100, and blocked with a BSA mixture. The samples were then stained with antibodies against human octamer‐binding transcription factor 4 (OCT4; R&D systems, 1:200), stage specific embryonic antigen 1 (SSEA1; R&D systems, 1:200), cluster of differentiation 31 (CD31‐FITC; Miltenyi Biotec, 1:100), CD34 (CD34‐PerCP, BioLegend, 1:100), human leukocyte antigen ‐ DR isotype (HLA‐DR, HLA‐DR‐APC, BioLegend, 1:100), CD73 (CD73‐PE, BioLegend, 1:100), CD90 (CD90‐APC, BioLegend, 1:200), CD105 (CD105‐FITC, BioLegend, 1:150), integrin α5 (Santa Cruz, 1:200), integrin α11 (Abcam, 1:200), integrin β1(Abcam, 1:200), and integrin β5 (BioLegend, 1:200) for 30 min or 1 h at 4 °C. Samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor‐488 or 594 conjugated goat antirabbit (Abcam, 1:400), Alexa Fluor‐488 or 594 conjugated goat antimouse (Abcam, 1:400)) for 30 min at 4 °C. The corresponding mouse/rabbit isotype antibodies (Abcam, 1:200) were used as controls. Cell immunotypes were determined with the Accuri C6 flow cytometer (BD Biosciences) and the percentage of expressed cell surface antigens was calculated for 10 000 gated‐cell events.

Mouse platelets were isolated as reported previously12 (link). Briefly, mice were anaesthetized by an intraperitoneal injection of Ketamine 100 mg/kg and Xylazine 10 mg/kg (AniMedica). Blood drawn directly from the heart was immediately collected in ethylenediaminetetraacetate (EDTA) tubes (S-monovettes, Sarstedt, Germany). Immediately after isolation, 5 μl EDTA blood was mixed with 95 μl FACS buffer (2 mM EDTA, 0.5% FCS ad 100 ml PBS, pH 7.1) and 16 μg/ml Thiazine Red and incubated for 3 hours at 4 °C. Then the cells were lysed using Miltenyi red blood cell lysis buffer, centrifuged and the pellet dissolved in 100 μl FACS flow. Some samples were incubated just prior the lysis with 5 μl of IgG1-FITC control (Miltenyi 130-089-867), CD62P-FITC (BD Biosciences, Heidelberg, Germany, Cat: 561923), CD31-FITC (Miltenyi 130-097-424) or CD61-FITC (Miltenyi 130-098-722) for 15 min at room temperature. FACs analysis was instantly performed with a BD FACScan. For the aggregation assays, isolated platelets were incubated with or without 2 mM CaCl₂ for 20 min at 37 °C, then fixed with 4% PAF, and incubated with 16 μg/ml Thiazine Red for 2 hours at 4 °C, cells were centrifuged and visualized under the microscope or in the FACS.

The number of circulating EPCs was assessed by FACS analysis by staining 1 million cells for two-color FACS analysis employing the following monoclonal antibodies: fluorescein isothicyanate-anti-CD34 (IQ products) and allophycocyanin-anti VEGF-receptor 2 (KDR, R&D systems). The various EPC phenotypes assessed were CD34+ and CD34+KDR+.
Late outgrowth EPC were characterized by the expression of several EPC markers: CD34-PE, KDR-APC, CD31-FITC and CD133- biotinylated (Miltenyi biotec), and secondary Rabbit anti mouse PerCP (Santa Cruz).