Egm2 bullet kit cc 3162

Human umbilical vein endothelial cells were cultured at 37^°C in 5% CO₂ in EGM-2 BulletKit CC-3162 (Lonza Walkersville Inc., MD). The cells were expanded in T-75 flasks between 5 and 7 passages; the culture media were changed every other day. Before seeding onto vascular grafts, cells were harvested using 0.25% trypsin, and 150,000 cells were added per graft and incubated in static culture for 4 days. In preliminary experiments, several different densities of cells were tested. The current density was found to be optimum in terms of favorable viability of cells and incubation period.

Immortalized human mammary epithelial (HMLE) cells expressing empty vector pWZL (HMLE-vector), Snail (HMLE-Snail) or Twist (HMLE-Twist), and RAS-transformed HMLE (HMLER) cells were maintained as previously described [37 (link), 51 (link)]. HMLE cells were transduced, as previously described [39 (link)], with expression constructs encoding green- or red-fluorescent protein (pMIG or RFP/luciferase, respectively), for use in tube-formation assays. MCF-7, MDA-MB-231, and SUM159 human breast cancer cells were cultured in cell-specific medium as previously detailed [28 (link)]. HMLE-Snail or MDA-MB-231 cells, transduced with shRNA targeting either firefly luciferase (shControl) or FOXC2 (shFOXC2), were previously described [28 (link), 29 (link)].
Cells were treated with vehicle (dH2O) or 100 μM desferrioxamine (DFX) for 24 hours prior to processing for immunofluorescence. For the induction of endothelial differentiation, cells were seeded at a density of 6000 cells/well and cultured for 8–10 days in endothelial cell growth medium (EGM-2 BulletKit™, CC-3162; Lonza Inc, Rockland ME, USA), supplemented with 2% fetal bovine serum and VEGF (designated EGM-2), as previously described [62 (link)].