Lipofectamine® 2000 Transfection Reagent

DNA transfection Mammalian cells - Primary cells Rat dermal fibroblasts (rDF)

Experiment
DNA transfection Mammalian cells - Primary cells Rat dermal fibroblasts (rDF)
Product
Lipofectamine® 2000 Transfection Reagent from Thermo Fisher Scientific
Manufacturer
Thermo Fisher Scientific

Protocol tips

Protocol tips
Do not add antibiotics to the Transfection Medium

Publication protocol

After cell isolation, four common non-viral transfection methods were used for the transfection of rat dermal fibroblasts: 1) Lipofectamine 2000 (Invitrogen, California, USA), 2) Jet PEI (Polyplus-transfection SA, Strasbourg, France), 3) Calcium Phosphate Transfection Kit (Invitrogen, California, USA) and 4) Transfection with the Nucleofector apparatus (later in the text referred as nucleofection) by using the Nucleofactor Kit for primary mammalian fibroblasts as described by the manufacturer (Lonza, Cologne, Germany). In addition, the modified nucleofection method was tested. Plasmid pmaxGFP (Lonza, Cologne, Germany) was used for all transfection experiments. Transfection efficiencies were monitored by GFP fluorescence using flow cytometry (Cytomation MoFlo® Flow Cytometer, Dako, Denmark). The transfection protocols were as follows:

Lipofectamine 2000: 0.2 million cells were seeded one day before transfection in one well of a 24-well plate in 1 ml cell culture medium. The cells were transfected upon reaching the confluence of 80-90%. Medium was changed short time before the transfection. Two mixtures were prepared. One contained 4 µg GFP and 50 µl DMEM, and the other 2 µl Lipofectamine 2000 and 50 µl DMEM. They were incubated at RT (room temperature) for 5 min. Subsequently, both solutions were thoroughly mixed, followed by incubation at RT for 20 min. 100 µl of the complete solution was added into the well with cultured primary fibroblasts and incubated for 4 hours in the incubator under standard conditions (37°C, 5%CO2). After incubation time elapsed, the medium containing the transfection solution was discarded and the fresh cell culture medium added to the cells. The transfection efficiency was measured after 48 h.

Jet PEI: 0.1 million cells were seeded one day before transfection in one well of a 24-well plate in 1 ml cell culture medium. The cells were transfected upon reaching the confluence of 80-90%. 1 µg pmaxGFP and 2 µl Jet PEI were resuspended in 100 µl of 150 mM NaCl and incubated for 15 min. The mixture was then added to the plated fibroblasts and incubated for 4 hours in the incubator under standard conditions. After incubation time elapsed, the medium containing the transfection solution was discarded and the fresh cell culture medium was added to the cells. The transfection efficiency was measured after 48 h.

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Manufacturer protocol

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