Penicillin streptomycin l glutamine heparin

Human ECFCs were isolated from neonatal cord and peripheral blood and their distinct endothelial phenotypes were verified by flow cytometry as previously described (see Supporting Information Fig. S1) (Hofmann et al., 2009; 2012,; Reinisch and Strunk, 2009 (link, link, link); Reinisch et al., 2009 (link)). HUVECs were obtained from Lonza (Basel, Switzerland). ECFC and HUVECs were grown in endothelial growth medium-2 (EGM-2) (Lonza) containing 2% FBS and 1% penicillin/streptomycin/L-glutamine/heparin (Life Technologies, Carlsbad, CA, USA) and EGM-2 growth factor supplements (composed of bFGF, IGF-2, EGF, VEGF, ascorbic acid, hydrocortisone). Ovarian carcinoma cell lines OVCAR-3 (American Type Culture Collection, Manassas, VA, USA), OVCAR-5 (kindly provided by the Cell Culture Core, Vascular Biology Program, Boston Children's Hospital, Boston, MA, USA) and COV-362 (Sigma Aldrich, St. Louis, MO, USA) were grown in DMEM containing 10% FBS.

Capillary-like network formation of ECFC, isolated from three different donors, plated on growth factor-reduced Matrigel® (BD, Biosciences, San Jose, CA, USA) was performed according to the instruction manual included in the purchase of Matrigel. The influence of LPI and different endocannabinoid receptor antagonists was tested in growth factor-reduced medium [EBM-2 (Lonza) containing 2% FBS and 1% penicillin/streptomycin/L-glutamine/heparin (Life Technologies) without the addition of EGM-2 growth factor supplements]. Network formation (14–16 h) was documented with a Nikon SPOT camera on a Nikon microscope (Nikon, Amsterdam, The Netherlands). Branch points were counted after 16 h by ImageJ [National Institutes of Health (NIH), Bethesda, MD, USA]. Nine independent experiments per group were performed in triplicate.

ECFCs from three different cord blood donors, HUVECs and peripheral blood ECFC were seeded in 24-well plates (Nalge Nunc, Rochester, NY, USA) in EGM-2 at a density of 3000 cells/cm² and allowed to adhere for 24 h. Subsequently, cells were subjected to growth factor-reduced medium [EBM-2 (Lonza) containing 2% FBS and 1% penicillin/streptomycin/L-glutamine/heparin (Life Technologies) without the addition of EGM-2 growth factor supplements] with or without different concentrations of LPI (Sigma) and/or endocannabinoid receptor antagonists: CID16020046 (Tocris Bioscience, Northpoint, Avonmouth, Bristol, UK) and AM251, SR144528 (both Cayman Chemical Europe, Tallinn, Estonia). A 30 min treatment with 10 μM U0126 (Cell Signaling, Cambridge, UK) was also tested. After 48 h, treated cells were harvested and the cell number was counted by a Casy cell counter (Roche, Mannheim, Germany). Nine independent experiments per group were performed in triplicate.