*2.7. Determination of Transfection E*ffi*ciency*

Lipofectamine™ 2000 transfection reagent was commercially obtained from Life Technologies, USA. The other transfection reagent NKS11, a new lipoid, was synthesized by others. In a safety test, the NKS11 formulation was proved to be nontoxic to Balb/c mice by weight measurement and health observation.

One day before transfection, adherent NIH3T3 cells (1.00 <sup>×</sup> <sup>10</sup><sup>5</sup> cells/well) in 1 mL growth medium without antibiotics were plated in a 12-well format so that cells would be 90–95% confluent at the time of transfection. GFP (green fluorescent protein) plasmids (1.6 μg/well, pRK-9-Flag-EGFP, 5520 bp, Addgene, USA) were transfected into the cells to determine the transfection efficiency. The medium was changed after 4–6 h. The growth medium was removed and the cells were washed with phosphate buffered saline (PBS), followed by trypsinizing the cell pellet for 5 min using trypsin (100 μL/well) at 1, 2, and 3 days post transfection, respectively. The cell pellet was resuspended with 1 ml growth medium and cell number was counted using a hemocytometer under the fluorescence microscope.

By the data we obtained, the transfection efficiency increased during the period 1, 2, and 3 days post transfection, and then saturated at the 3rd day. The percentages were about 14.3%, 21.4%, and 21.4% using lipofectamine. The results revealed that the highest transfection efficiency for plasmids in NIH 3T3 cells was about 20–25% and it took about 2–3 days for the plasmids to transfect into cells completely. NKS 11 showed almost the same efficiency for transfection in NIH 3T3 cells.
