Bs 1 lectin binding

This study was approved by the Ethics Committee of First Affiliated Hospital of Jinan University. All donors provided written informed consent. Human late EPCs were purified from 20 healthy individuals and cultured following previously described methods.
³¹ (link) Briefly, peripheral blood mononuclear cells (PBMNCs) were maintained in endothelial cell basal medium‐2 (Lonza, Basel, Switzerland) supplemented with endothelial growth medium‐SingleQuots (Clonetics, San Diego, CA) in fibronectin‐coated 6‐well plates. After 4 days of culture, nonadherent cells were removed. The adherent cells were cultured for 28 days, with the medium being refreshed every 7 days. After 28 days of culture, human late EPCs were characterized by a double‐positivity for BS‐1 lectin binding (0.01 mg/mL; Sigma‐Aldrich, St. Louis, MO) and Dil‐acetylated low‐density lipoprotein uptake (0.02 mg/mL; Invitrogen, Carlsbad, CA). Cultured EPCs were incubated with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindo‐carbocyanine‐acetylated low‐density lipoprotein for 2 hours in a cell incubator. Subsequently, cells were washed and fixed with 4% paraformaldehyde for 15 minutes and incubated with FITC‐BS‐1 lectin for 1 hour. Plates of cells were again washed and incubated with DAPI nuclear counterstain. Double‐positive cells were observed with a fluorescent microscope (×200 magnification; Olympus, Tokyo, Japan).

EPCs were isolated and cultured as previously described in detail [24 (link),25 ]. After 4 days culture, non-adherent cells were removed by thoroughly washing with endothelial cell basal medium-2 (EBM-2) (Lonza, Swiss). Medium was changed daily for 7 days, and then every 3 days was changed. After 4 weeks' culture, late EPCs were defined as cells dually positive for 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanine (DiI)-acetylated low density lipoprotein (acLDL) uptake (0.02 mg/ml; Invitrogen, Carlsbad, CA, USA) and BS-1 lectin binding (0.01 mg/ml; Sigma-Aldrich, St. Louis, MO, USA). Endothelial markers of cultured EPCs were also examined by flow cytometry analysis using CD31 (BD Pharmingen), von Willebrand factor (vWF) and kinase-insert domain receptor (KDR) (R&D Systems Inc) as previously described [26 (link),27 (link)]. Based on the isolation and cultivation protocol, the adherent mononuclear cells were identified as late EPCs, and then were used for the following experiments.