Vasculife engs medium

Human umbilical vein endothelial cells (HUVECs) were employed as a model of neutrophil TEM. Lifeline Cell Technology was used to harvest HUVECs [28 (link),29 (link)]. The cells were grown in VascuLife^® EnGS medium (Lifeline Cell Technology, Frederick, MD, USA; LS-1019; including 5 ng/mL rhEGF, 0.2% EnGS, 0.75 U/mL, 2% FBS, 10 mM L-glutamine, 1 μg/mL hydrocortisone, and 50 μg/mL ascorbic acid). The cell cultures were incubated in room air with 5% CO₂ at 37 °C and 95% humidity and were expanded by brief trypsinization with 0.25% trypsin in PBS containing 0.025% ethylenediaminetetraacetic acid (EDTA). The experiments were conducted on passage 3–5 HUVECs.

Human umbilical vein endothelial cells (HUVEC), human dermal microvascular endothelial cells (HDMVEC) and human aortic endothelial cells (HAEC) were purchased from Lifeline Cell Technology and cultured in VascuLife EnGS Medium (Lifeline Cell Technology) containing cell-specific growth supplement. Cells from passages 2–6 were used for experiments. Human brain microvascular endothelial cells (HBMEC) was purchased from Lonza and cultured in VascuLife Basal Medium (Lifeline Cell Technology) supplemented with growth factors for human microvascular endothelial cells. Cells were maintained at 37°C and 5% CO₂ with humidity. Cell confluence was visually determined when cells were in contact and the entire culture surface had no visible space among individual cells for at least 48 h. For autophagy induction, cells were starved with HBSS for 1 h at 37°C, or 250 nM rapamycin in complete medium for 12 h at 37°C.

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).