Transfection experiments Sample Clauses

Transfection experiments. One day before the transfection experiment, cells were seeded into 96-well plates at a density of 1x104 cells per well in standard cell culture media (plating volume 100 µl) and cultured overnight at 37°C. Just before transfection, the medium was removed and 100 µl of HBSS buffer was added to each well. After 30 min HBSS was removed and an equal volume of the complexes LD, LpegD and LPD of different charge ratios (0.5, 1, 2, 3, 4) were added to each well in filtered HBSS. The final DNA concentration was 0.2 µg per well. The cells were transfected for 6 h before removal of the complexes and each well was then supplied with 100 µl standard cell culture medium. Cells were incubated for 48 h to allow gene expression to proceed. The negative control group consisted of naked DNA alone in HBSS. Lipofectamine (0.05 µl/well) complexed to DNA (0.2 µg/well) was used as a positive control because of its reported high transfection activity. This commercial transfection reagent is suitable for in vitro transfection of suspended or adherent cells and it has been optimised for the delivery of both DNA and siRNA in a wide range of cell lines including Calu-3.
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