Basic fibroblast growth factor (bfgf)

For the determination of sphere forming activity, adherently growing NSCLC cell lines H1299 and A549 were harvested and re-suspended in sphere-forming medium, containing 0.4% bovine serum albumin (Sigma-Aldrich), 10 ng/ml Epidermal Growth Factor (Sigma-Aldrich), 20 ng/ml Basic Fibroblast Growth Factor (VWR, Søborg, Denmark) and, in the case of H1299, 0.25 μg/ml human insulin (Sigma-Aldrich). To ensure a single cell suspension, the cells were filtered through 20 μm Steriflip filters (Millipore, Hellerup, Denmark), before being counted on a Cedex XS cell counter (Roche). The cells were seeded at densities of 250, 500, 1000, 2000, 4000 or 8000 cells/well in 150 μl medium in Corning^™ Ultra-Low Attachment 96-Well Plates (Sigma-Aldrich). At day 6, spheres were counted under the microscope. Three pictures were taken per well, and the diameter of spheres was determined with the Cell^F program (Olympus Europe). For the sphere-based viability assay, 20μl CellTiterBlue^™ (Promega, Nacka, Sweden) was added per well, and the plate incubated for 23 hours prior to viability readout with a Wallac Victor3 1420 Multilabel Counter (Perkin Elmer, Skovlunde, Denmark) at 560_ex/590_em nm. Experiments were performed with 10 wells per setting, and repeated three times.