Cell Culture Protocols for Various Cell Lines

Human umbilical vein endothelial cells
(HUVECs) were obtained from TCS Cell Works (Buckingham, UK) and were
cultured on tissue culture-coated flaks in endothelial basal medium
(EBM; Cambrex BioScience Ltd., Nottingham, UK) supplemented with gentamycin–ampicillin,
epidermal growth factor, and bovine brain extract (Singlequots; Cambrex)
and 10% fetal bovine serum (FBS) (complete EBM). For experimental
purposes, fully confluent HUVECs at passages 1–3 were preincubated
overnight with 1% FBS in EBM prior to addition of factors and other
treatments. DU145/cells.Ad.NRP1 cells: DU145 prostate cancer cells
were from ATCC and infected with an adenovirus expressing WT NRP1
as previously described^{27 (link)} (cells were grown
in 10% FCS in DMEM). A375P melanoma cells were from ATCC. Cells were
grown in 10% FCS in DMEM.
Human dermal fibroblasts (HDF) were
obtained from TCS cells Works Buckingham, UK) and were cultured in
Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco).
For experimental purposes, fully confluent HDF at passages 1–3
were preincubated overnight with serum-free DMEM prior to other procedures.
GL261 cells were purchased from ATCC and cultured as described.^{44 (link),45 (link)}