12 well transwell

Migration assays were performed using 12-wellTranswell (Corning, USA) with 10⁵ cells in DMEM on the upper chamber and DMEM/10% FBS in the lower chamber. 16 hrs after seeding, cells in the upper chamber were fixed, stained with DAPI, and photographed. Ex vivo metastasis assay was performed according to a published protocol.^{20 (link)} Briefly, 2×10⁵ cells were IV injected into the FVB/N mouse via tail vein. The mice were euthanized by CO2 inhalation within 15 minutes of tumor cell injection. Mouse lungs were exposed, infused with well-mixed culture medium/agarose solution, then removed and placed in a cold solution of PBS to solidify the agarose/medium solution. Transverse sections were made from each lobe using a #21 scalpel blade, were placed on a single sterile Gelfoam (Pfizer-Pharmacia & Upjohn Co.) section in a 6-cm tissue culture dish with culture medium. Lung sections were incubated at 37°C in humidified conditions of 5% CO2. Fresh culture medium was replaced and lung tissue sections were turned over with a sterile iris thumb forceps every other day. GFP+ tumor cells were photographed using a Zeiss Stemi SV 11 Stereo-Microscope equipped with Kramer Scientific Quad-Fluorescence illumination and counted without prior knowledge of the genotype of the cell. All animal studies were approved by the Animal Care and Use Committee of UT Southwestern.