Forty-eight hours after viral transduction, hygromycin B (Invitrogen) was included in the medium at a concentration of 25 ��g/ml for 2 weeks. Following drug selection, individual colonies were picked and expanded into cell lines. To generate drug-resistant cancer cells, Hepa1�C6 cells were http://www.selleckchem.com/products/chir-99021-ct99021-hcl.html transfected with a neomycin resistance gene and a green fluorescent protein gene with the Effectene Transfection Reagent kit (Qiagen). Forty-eight hours after transduction, neomycin (Invitrogen) was included in the culture medium at a concentration of 100 ��g/ml, and individual colonies were picked at day 14 and expanded into cell lines. Cell Fusion For polyethylene glycol fusions, cells of each type (generally 5 �� 106) were combined in serum-free Glasgow minimum essential medium in a conical tube and pelleted.

After the supernatant was aspirated, the pellet was broken by gentle tapping, and 1 ml of 50% w/v polyethylene glycol 1500 (Roche Diagnostics, Basel, Switzerland) prewarmed to 37 ��C was gently added. Cells were incubated in the 50% polyethylene glycol solution for 1 min with occasional stirring. Then, 1 ml of medium was added over a period of 1 min. Subsequently, an additional 3 ml of medium was added. Cells were centrifuged, and the supernatant was discarded. The pellet was resuspended in complete ES cell medium and plated. Selection was applied after 48 h using hygromycin (200 mg/ml) and neomycin (100 mg/ml). Fourteen days following drug selection, single colonies were picked and expanded under standard conditions. Karyotype Analysis A 25-cm flask at 60% cell confluence was treated with 0.

04�C0.1 ��g/ml colchicine for 3 h. Cells were recovered by trypsinization and treated with a hypotonic (0.56% w/v) KCl solution for 15 min. The cells were centrifuged at 500 rpm, fixed by washing three times in fresh fixative (3:1 methanol:acetic acid), and dropped onto cold, clean glass slides. The slides were air-dried, stained with 4��,6-diamidino-2-phenylindole, and observed under a microscope. Fluorescence-activated Cell Sorting For analysis of DNA content, cells in a 10-cm dish were trypsinized, washed in phosphate-buffered saline (PBS), and fixed with 70% ethanol at 4 ��C for 30 min. RNase A was added to 500 ��l of PBS at a final concentration of 20 ��g/ml. Cells were incubated in this solution at 37 ��C for 30 min and then centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the cells were incubated Brefeldin_A with propidium iodide in 500 ��l of PBS at a final concentration of 50 ��g/ml in the dark at room temperature for 30 min and then centrifuged at 1500 rpm for 5 min. The supernatant was discarded again, and the cells were resuspended in 0.5 ml of PBS. The stained cells were analyzed with the FACSCalibur system (BD Biosciences).