SatisFection™ Transfection Reagent From Stratagene

Stratagene claims that SatisFection™, a new cationic polymer, is suitable for transfection of a number of different cells lines, including difficult to transfect cells and primary cells with observed transfection efficiencies ranging from 10 to 60% (depending on the cells used). Since only small amounts of the reagent are used, the price is highly competitive.

I have not used SatisFection™ for many different cells, but I know from my experiences that it works great with the mouse myeloma cell line NSO. I transfect these cells with various antibody genes cloned into a human IgG1 expression vector for antibody production. For stable transfection, 0.5 x 10⁶ cells/transfection are seeded into a 6 well plate the day before transfection and fed with 4 ml medium containing serum and antibiotics. (Stratagene has a note in the protocol that the efficiencies are better when transfection of adherent cells is performed in serum-containing medium compared to serum-free medium.) The cells should grow to a 60 to 80% confluent layer. The day of the transfection, plasmid DNA carrying the cloned heavy and light chain antibody rearrangements (2 µg DNA each) is mixed with antibiotic-free, serum-free cell culture medium at room temperature (total volume 100 µl); in a separate tube, 3 µl of the transfection reagent stock solution is gently mixed with antibiotic-free, serum-free cell culture medium at room temperature (total volume 100 µl). Here the medium has to be really serum-free. The optimal DNA to reagent ratio can be optimized for highest efficiencies and low levels of cytotoxicity. I am using 1.5 µl reagent per µg DNA, and in double transfections (heavy and light chain immunoglobulin rearrangement at once), 1 µl reagent per µg DNA, with good results. Then the transfection reagent/medium mixture (100 µl) is added drop wise into the DNA/medium mixture (100 µl) and the combined solutions are incubated for 15 minutes at room temperature. The transfection reagent/DNA/medium mixture (200 µl) is added drop wise onto the cells in 4 ml complete medium and mixed with the cell culture by gently rocking the multi-well dish. For stable transfection, the medium is replaced with the respective selective medium, depending on the selective markers present on the plasmids used for cloning and transfection (e.g. Hygromycin B, G418, HAT) after a 24 h incubation period. It is convenient to plate the cells in this step into 96-well plates because positively transfected cells grown in individual wells are easier to handle for testing than bulk culture. I usually change the selective medium seven days after transfection and incubate for another three to five days and then proceed to test all wells showing cell growth by ELISA. The whole time frame for a stable transfection is 10 to 14 days. After testing of the transfected cells, one should select the cell lines showing the highest level of gene expression and clone them by limiting dilution. At this time point, it is good to freeze an aliquot of the cells to secure the experiment.

Personally, I have used a number of different reagents and methods to transfect eukaryotic cells, starting from the early days with DNA/CaCl₂ coprecipitation, and I really like this new reagent. My efficiencies for this particular cell line, which is not noted in the company information sheet, are around 30% for single transfection and around 15% when I perform transfection with two different plasmids at once.