Egmtm 2 media

Human lung epithelial adenocarcinoma cells (A549, ATCC), human embryonic kidney cells (293T, ATCC), mouse lung epithelial cells (LA-4, ATCC) and mouse endothelial cells (MS1, ATCC) were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Denville Scientific) and penicillin/streptomycin (Pen/Strep, 100 units/mL, Corning). Human lung microvascular endothelial cells (HMVEC, Lonza) were maintained in EGM^TM -2 media according to the supplier’s instructions (Lonza). Madin-Darby Canine Kidney (MDCK, ATCC) cells were maintained in Minimum Essential Medium (MEM; Lonza) supplemented with 10% FBS and Pen/Strep (100 units/mL). THP-1 cells (ATCC) were maintained in RPMI media (Gibco) supplemented with 10% FBS and Pen/Strep (100 units/mL). Bone marrow derived dendritic cells (BMDC) were prepared from the tibia and femur of C57BL/6J mice.

HUVEs and EGM^TM-2 media (Lonza, Walkersville, MD, USA) was purchased. HUVECs (3×10⁴ in 200 μl of EGM-2 medium) were seeded onto 0.1% gelatin-coated inserts in the transwell chamber (8-μm pore size and 6.5-mm diameter) and allowed to form a monolayer for 48 h. The endothelial culture medium was removed from each insert and CFSE-labeled cancer cells (1×10⁵ cells in 200μl of serum-free medium) were added on top of the HUVEC monolayer. Cancer cell culture medium containing 10% FBS was added to the lower chamber. After incubation for 48 or 72 h, non-migratory cells were wiped off with cotton swabs and the membrane was washed with PBS. Finally, the membranes were removed from the transwell insert, mounted onto slides and observed under a fluorescence microscope at ×10 magnification.