Fetal calf serum fbs

SK-BR-3, BT-474, MDA-MD-468, MDA-MB-453 and HCC1419 cell lines were obtained from American Type Culture Collection (Rockwell, MA).The JIMT-1 cells were a gift from Pravin Kaumaya, (The Ohio State University, Columbus OH). SKBR3 cells were cultured in McCoy’s 5A Media (Gibco) supplemented with 10% v/v fetal calf serum (FBS; BioWhittaker), 100 units/ml of potassium penicillin and 100 μg/ml of streptomycin sulfate (BioWhittaker). BT-474 cells were cultured in HybriCare (ATCC), supplemented with 10% v/v FBS, 100 units/ml of potassium penicillin and 100 ug/ml of streptomycin sulfate. MDA-MB-468, MDA-MB-453 and HCC1419 cells were cultured in RPMI-1640 (BioWhittaker), 10% v/v FBS, 100 units/ml of potassium penicillin and 100 ug/ml of streptomycin sulfate (BioWhittaker), 2mmol/L glutamine (BioWhittaker), 1mmol/L sodium pyruvate (BioWhittaker), and 1% non-essential amino acids (BioWhittaker). All cells were maintained in culture at 37°C in 5% CO₂.

HUVECs were purchased from BD Biosciences (San Jose, CA, USA), and cultured on gelatin-coated T-75 tissue culture flasks with supplemented M199 medium (Life Technologies, Carlsbad, CA, USA). M199 was supplemented with 1 % (v/v) 0.2 M L-glutamine, 1.5 % (v/v) 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid from Lonza Walkersville, Walkersville, MD, USA), 1.8 % PSG (penicillin-streptomycin-glutamine from Lonza Walkersville, Walkersville, MD, USA), 15 % (v/v) fetal calf serum (FBS), sodium bicarbonate (Lonza Walkersville, Walkersville, MD, USA), and heparin salt (Fisher Bioreagents, Fair Lawn, NJ, USA). Endothelial cell growth supplement (Alfa Aesar, Ward Hill, MA, USA) was added to the supplemented M199 to achieve a final concentration of 40 μg/ml. Full media was stored at 4 °C for use up to 4 weeks. HUVECs were grown to 80 % or greater confluency and were passaged using a 50:50 mixture of trypsin-versene and HBSS (Hank’s Balanced Salt Solution from Lonza Walkersville, Walkersville, MD, USA) before being used in experiments. Only cells between passages 3 and 6 were used.