Three agents were used for transfection: MWCNT, Lipofectamine 2000 (Thermo Fischer Scientific), and X-tremeGENE HP Reagent (Roche, Basel, Switzerland). All experiments were performed in triplicate. In a microfuge tube, 100 μL of MWCNT 0.25 mg/mL was added to 300–500 ng of plasmid DNA (pDNA) and kept in an ultrasonic bath at 40 kHz and 120 W for 30 min at 4°C, then kept for 10 min at 4°C. The MWCNT-pDNA complex was then added to the well containing the cells to be transfected into a final volume of 2 mL, homogenized by rotational movement, and incubated at 37°C in a humidified atmosphere containing 5% CO2.
The X-tremeGENE HP Reagent (Roche) was used to transfect the cell lines according to the manufacturer’s specifications with modifications like those used for Lipofectamine 2000 (Thermo Fischer Scientific). Six microlitres of each reagent were incubated with 1 μg of pDNA in 100 μL of DMEM supplemented with 1 mM sodium pyruvate and 0.1 mM non-essential amino acids for 10 min and added to the cells. Twenty-four hours before the transfection procedure, 1 × 104 cells/well were seeded in a 6-well plate corresponding to 70% confluency at the beginning of the transfection. DMEM supplemented with 1 mM sodium pyruvate and 0.1 mM non-essential amino acids was used for culture.
The X-tremeGENE HP Reagent (Roche) was used to transfect the cell lines according to the manufacturer’s specifications with modifications like those used for Lipofectamine 2000 (Thermo Fischer Scientific). Six microlitres of each reagent were incubated with 1 μg of pDNA in 100 μL of DMEM supplemented with 1 mM sodium pyruvate and 0.1 mM non-essential amino acids for 10 min and added to the cells. Twenty-four hours before the transfection procedure, 1 × 104 cells/well were seeded in a 6-well plate corresponding to 70% confluency at the beginning of the transfection. DMEM supplemented with 1 mM sodium pyruvate and 0.1 mM non-essential amino acids was used for culture.