DNA is first mixed with Enhancer and a buffer that provides optimal salt conditions for efficient DNA condensation. This step requires just 2-5 minutes. Effectene Reagent is then added and the mixture is incubated for 5-10 minutes to allow Effectene-DNA complexes to form. The complexes are mixed with growth medium (which can contain serum and antibiotics), and added directly to the cells. The cells are then incubated until harvested and analyzed for gene expression.
C6 glioblastoma cells (1 x 104) were transfected with either naked FITC-labeled CD44 antisense oligodeoxynucleotide (ODN) or with Effectene-ODN complexes. Intracellular accumulation and distribution of the transfected ODNs was analyzed 16 hours post-transfection by immunofluoresence. [A] Naked ODNs (2 µg). [B] ODNs (0.04 µg) complexed with Effectene Reagent. Transfected ODNs appear yellow in the photos. (Data kindly provided by G. Beutel and M. Schott, Institute of Neuropathology, Hanover Medical School, Hanover, Germany.)
The influence of serum and DNA quantity on transfection using Effectene Reagent was examined. COS-7 cells (2 x 104 per well) were seeded in 96-well plates one day before transfection. Cells were transfected using 0.01–1.0 µg beta-galactosidase-reporter plasmid and 0.08–8.0 µl Enhancer (DNA: Enhancer ratio of 1:8) and 2 µl Effectene Reagent, in either the presence or absence of serum. Each bar represents the average efficiency from 4 replicates 48 hours post-transfection.
Primary rabbit aortic smooth muscle cells were transfected with 0.4 µg of a green fluorescent protein reporter plasmid using Effectene Reagent in 6-well plates. Cells were viewed by fluorescence microscopy 24 hours after transfection. Approximately 40% of the cells were transfected, as determined by FACS analysis. (Data kindly provided by K. Veit, 2nd Medical Clinic, Dept. Clinical Pharmacology, Mainz, Germany.)
Transfection efficiencies using Effectene Reagent and two commonly used lipid-based reagents were compared. Murine teratocarcinoma F9 cells (5 x 105) were transfected in 6-well plates with a luciferase-reporter plasmid using optimized conditions based on the manufacturer's instructions for each reagent. Transfection efficiencies were determined by measuring luciferase activity 48 hours post-transfection, and are given as relative light units (RLU). (Data kindly provided by I. Clavereau, D. Petitprez, and I. Van Seuningen, Unité INSERM 377, Place de Verdun, Lille Cedex, France.)