Vasculife engs lifefactors kit

Prior to cell cultivation, electropolished/blue oxide or native Nitinol test specimen were preincubated for 10 min at 37 °C under soft agitation with 1-ml platelet-rich plasma (PRP). PRP was prepared from fresh human whole blood, which was anticoagulated with 3 IU/ml Heparin, by centrifugation for 10 min at 160 g and room temperature. After washing with PBS, each plate was put into a well of a 12-well plate and seeded with 120,000 human umbilical vein endothelial cells (HUVECs) in 1 ml VascuLife EnGS medium (Lifeline Cell Technology, USA) containing VascuLife EnGS LifeFactors Kit, 50 µg/ml gentamicin and 0.05 µg/ml amphotericin B (PAA Laboratories, Germany). The samples were incubated for 48 h at 37 °C and 5% CO₂. The HUVECs were isolated as previously described [14 (link)].
After incubation, the samples were washed with PBS, fixed using CellFix (BD Biosciences, Germany) and subsequently permeabilized in 90% ice-cold methanol. After washing, DAPI nuclear dye (300 nmol) was incubated for 3 min and the samples were investigated using a fluorescence microscope (Optiphot-2, Nikon, Germany) equipped with a DSLR remote control (Nikon 550 D).

HEK293 cells were cultivated in DMEM with high glucose (PAA Laboratories, Cölbe, Germany) supplemented with 10% FBS (Life Technologies, Darmstadt, Germany), 2 mM L-glutamine (PAA Laboratories), and 1% penicillin/streptomycin (PAA Laboratories). BJ human foreskin fibroblasts (Stemgent, Cambridge, USA) were cultivated in DMEM with high glucose containing 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin, and 30 mM HEPES (Life Technologies, Darmstadt, Germany). Human endothelial cells (ECs) were isolated as described previously [12 (link)] and cultivated in flasks precoated with 0.1% gelatin in Vasculife® EnGS EC culture medium (CellSystems, Troisdorf, Germany) containing VascuLife EnGS LifeFactors Kit, 50 mg/ml gentamicin, and 0.05 mg/ml amphotericin B (PAA Laboratories). Cells were kept at 37°C with 5% CO₂ and media was changed every 3 days. Cells were passaged using trypsin/EDTA (0.04%/0.03%, PromoCell, Heidelberg, Germany). BJ fibroblasts at passage 7-9, endothelial cells at passage 3-5 were used for all experiments.