Ulex lectin

EOCs and ECFCs were incubated with DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) (Invitrogen, Carlsbad, CA, USA) for 2 h followed by 4 % paraformaldehyde fixation and subsequent 1-hour incubation with FITC-labeled Ulex-lectin (Sigma-Aldrich, St-Louis, MO, USA). Nuclear staining was done with TO-PRO-3 (Invitrogen). Cells were visualized under an LSM 510 confocal microscope (Zeiss, Oberkochen, Germany) at 63× objective magnification [18 (link)].

CACs were the adherent mononuclear cells of a 4- to 7-day culture procedure (23 (link)). The number of CAC cells was assessed in 15 SCT and 14 control subjects. Mononuclear cells (1 × 10⁶) were plated on fibronectin-coated 24-well plates enriched with endothelial medium (basal medium with supplement pack, PromoCell, Heidelberg, Germany, supplemented with 20% fetal calf serum). The medium was changed after 72 and 120 h.
Counting was performed on day 5 in 15 randomly selected high-power fields (HPF, ×400) by fluorescence microscopy after incubation with acetylated-LDL (5 mg/L; Invitrogen, Carlsbad, CA, USA) and Ulex lectin (10 mg/L; Sigma, Saint Louis, MO, USA) for 4 h at 37°C. CAC cells were identified as cells with combined uptake of acetylated-LDL and Ulex lectin binding.