HUVEC Culture for Neutrophil Transendothelial Migration

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.