Cell Culture of Cancer and Endothelial Cell Lines

HT-29 colon cancer cell line and 2H-11 mouse endothelial cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), while HUVEC was purchased from Lonza (Basel, Switzerland). Cells were maintained in a complete medium, either Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS) (Corning, NY, USA), Penicillin (100 U/mL), Streptomycin (100 µg/mL) (Life Technologies) and 2 mM L-alanyl-L-glutamine dipeptide (GlutaMAX Supplement; Life Technologies) for HT-29 and 2H-11 or EBM™-2 Endothelial Cell Growth Basel Medium, supplemented with EBM™-2 Endothelial SingleQuots™ kit (Lonza) for HUVEC. Cells were grown under standard culture conditions (37 °C with 5% CO₂) and passaged no more than 5 times for HUVEC and no more than 20 times for HT-29 and 2H-11. All cell lines were mycoplasma-free, as determined using the MycoAlert™ Mycoplasma Detection Kit (Lonza) and/or a custom PCR-based assay, as described previously [11 (link),51 (link)].