Dii ac ldl

Cells were incubated with 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated LDL (Ac-LDL-DiI, Biomedical Technologies) as indicator of endothelial cells differentiation [16] (link), [17] (link). After fixation with 4% PFA, cells were counterstained with Hoechst 33258 nuclear and observed with a Zeiss microscope equipped for epifluorescence.

After 8 days in culture, the EPC-CFU cultures were treated with 0.4 µg/mL 1,1′-dioctadecyl-3,3,3′,3-tetramethyl-indocarbocyanine perchlorate-labeled acLDL (acLDL-DiI; Biomedical Technologies Inc., Stoughton, MA) for 1 h and fixed by application of 1 mL of 2% paraformaldehyde (PFA) (Affymetrix, Santa Clara, CA) for 1 h at room temperature. After washing with methylcellulose-PBS, the cultures were reacted with fluorescein isothiocyanate (FITC)-conjugated lectin from Ulex europaeus (UEA-I; Sigma-Aldrich) for 1 h at room temperature. The cultures were washed with PBS and then imaged using a confocal fluorescence microscope (Olympus).

After the induction of MSC differentiation into blood vascular cells, the cells were characterized as adherent cells that were double-positive for DiI-acLDL (Biomedical Technologies, USA) uptake and fluorescein isothiocyanate (FITC)-UEA-1 (Sigma) binding. Cell nuclei were counterstained using DAPI [51 (link)].

EPCs (10³ cells/well) were seeded on 1% fibronectin coated 24-well plates in EGM-2 Bullet Kit with 5% FBS (EGM-2 growth media, Lonza, Victoria, Australia) and allowed to attach following overnight incubation. For LDL uptake assay, cultured medium was replaced with 10 μg/ml of acetylated low density lipoprotein (Ac-LDL), labeled with 1,1′-dioctadecyl– 3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL, Biomedical Technologies), then cells were incubated for additional 4 h. After the end of the incubation period, the solution was aspirated and fresh growth medium was added before capturing the images using fluorescence microscopy (Zeiss AxioImager.2 microscope).

Cells were stained by periodic acid-Schiff (PAS) (Sigma-Aldrich, 395B) and DiI-ac-LDL (Biomedical Technologies, BT-902) following the manufacturer’s instructions. For the indocyanine green (ICG) (Sigma-Aldrich, I2633) uptake assay, the cells were changed to medium with 1 mg/mL ICG and incubated at 37 °C for 1 h. The cells were washed three times with PBS and imaged using an inverted microscope (Leica). For Oil red O staining, cells were washed three times with PBS and fixed with 4% PFA for 30 min. The ells were washed three times with PBS and then stained with Oil red O (Sigma-Aldrich, 00625) dissolved in isopropanol for 30 min. Then, the cells were washed twice with 60% isopropanol and imaged by inverted microscopy.

EPCs were prepared as previously described. [10] (link) Peripheral Blood Mononuclear Cells (PBMCs) were isolated from peripheral blood of healthy human volunteers as described by Sun et al by density-gradient centrifugation with Ficoll. Immediately after isolation, total MNCs (5*106 cells/mL medium) were plated on culture dishes coated with human fibronectin (Sigma) and maintained in endothelial basal medium (EBM, CellSystems) supplemented with EGM SingleQuots, VEGF (10 ng/mL) and 20% fetal bovine serum. The medium was replaced every 3 days. Adherent cells on 8 days of culture were stained by acetylated LDL and were labeled with DiI (DiI-acLDL, Biomedical Technologies) and fluorescein isothiocyanate (FITC)-labeled lectin from ulex europaeus (Sigma). Double-positive cells for DiI-acLDL and FITC-labeled lectin were identified as EPCs, as reported previously. All human researches were conducted in Changzheng Hospital. All procedure were permitted by the human volunteers with written consent and approved by the Committee on the Ethics of human Experiments of Changzheng Hospital.