Ulex europaeus agglutinin lectin

Detection of EPCs has been noted in seminal studies [18 (link), 25 (link)]. As mentioned, 100 μL peripheral blood was immunostained with monoclonal antibodies against human CD34 (Becton Dickinson, Franklin Lakes, NJ, USA, PerCP-conjugated) and KDR (Sigma, St. Louis, MO, USA), followed by a PE-conjugated secondary antibody. Isotype-identical antibodies acted as controls (Becton Dickinson). After incubation, cells were lysed, washed with PBS, and fixed in 4% paraformaldehyde. And then, the analysis of 60,000 events took place after exclusion of debris and platelets. The number of circulating EPCs was judged by the ratio of CD34+KDR+ cells per 100 peripheral blood mononuclear cells (PBMNCs). To confirm the EPC phenotype, the attached cells were incubated with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate-labeled acetylated LDL (DiI-acLDL, 10 μg/mL, Molecular Probes) at 37°C for 1 h. Then, the cells were fixed with 4% paraformaldehyde for 30 min at 37°C and incubated with FITC-labeled Ulex europaeus agglutinin (lectin, 10 μg/mL, Sigma) for 4 h at 37°C. After being stained, the samples were observed with a phase-contrast fluorescent microscope by two independent observers blinded to the study.

After 7 days culture, the plated EPCs on the cell culture flasks were incubated with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanine (DiI)-acetylated low density lipoprotein (acLDL) (DiI-acLDL, 10 μL/mL, Molecular probes) at 37°C for 1 h and they were incubated with FITC-labeled Ulex europaeus agglutinin (lectin, 10 μg/mL, Sigma). After staining, the samples were observed under a phase-contrast fluorescence microscope (magnification, ×200). Cell demonstrating double-positive fluorescence were identified as differentiating EPCs.