Use of flow cytometry to rapidly optimize the transfection of animal cells

Plasmid transfection is the first step in the generation of stably transfor med animal cells and is also a useful tool for analyzing transient gene exp ression. Maximizing the transfection efficiency and expression level from t he introduced plasmid is critical to the success of these processes. By mea ns of lipid-mediated transfection, a plasmid vector expressing the green fl uorescence reporter protein has been coupled with flow cytometry to conveni ently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rap id and simultaneous quantification of transfection efficiency and heterolog ous protein expression level per cell. Using this technique, we developed a n optimized transfection protocol for insect cells by investigating followi ng parameters: lipid incubation time, lipid/DNA mixture incubation time, li pid and DNA concentration, incubation vessel and transfection duration. Fol lowing optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.