Epics xl system 2

The recombinant lentivirus was produced by transfection of 293FT cells with the lentiviral expression vectors, pCMV-VSV-G-RSV-Rev, and pCAG-HIVgp, using Lipofectamine 2000 reagent (Invitrogen). After 72 h, the medium was collected, and 1 × 10⁵ of A549 or HFL-1 cells were infected with 500 μL of medium containing lentiviruses. For double knockdown of TGF-β1 and TGF-β2, 250 μL of each lentivirus-containing medium were used. Infection efficiency was assessed by measuring the percentage of EmGFP-positive cells via flow cytometry (EPICS XL System II; Beckman Coulter, Brea, CA), and knockdown efficiency of target gene was analyzed using an enzyme-linked immunosorbent assay (ELISA).

HCT116 cells were seeded at a density of 5.0 × 10⁴ cells/mL in 60-mm dishes and incubated in a humidified atmosphere of 5% CO₂ at 37 °C for 24 h. DMPC (0.1–0.6 mM) and HLs (0.1–0.6 mM, on the basis of the DMPC concentration) were added to each dish and the dishes were further incubated for 48 h. The cells were harvested by centrifugation and then resuspended in phosphate-buffered saline (PBS(−)) containing 1 mg/mL RNase, 0.1% Triton X-100, and 40 μg/ml propidium iodide (PI, Molecular Probes, Eugene, OR) in a dark room. The percentage of apoptotic cells was analyzed using a flow cytometer (Epics XL system II, Beckman Coulter, CA).