Dnasefree water

Poly(ethyleneimine) (Sigma Aldrich, MW 800, cat. no. 408719) was dissolved in water to prepare a stock solution (90 mg ml -1 ). Aqueous PEI was sonicated in a bath for 15 min. FND stock solution (2 mg ml -1 ) was added to the PEI solution (1 : 1 vol : vol) and sonicated with a probe (400 W, 15 min) under water cooling. The solution was washed with deionised water and centrifuged at 14 500g for 15 min (3 times) to remove residual PEI. The pellet was then redispersed in deionised water to a final concentration of 0.48 mg per ml of FND-PEI complex (based on FND concentration).
DNA (160 ng) was dissolved in 5 µl water and sonicated in a bath cooled with ice for 1 h. This solution was then added to a 25 µl FND-PEI complex (0.48 mg ml -1 , containing 12 µg FND) followed by sonication for 2 h. The FND-PEI-DNA complex was isolated by centrifugation at 14 000g for 15 min, and the pellet was resuspended in an appropriate amount of DNAsefree water (Qiagen) to obtain a stock solution of 2 mg per ml FND-PEI-DNA.

The equivalent of 10 5 cells IC-21 maintained in RPMI1640 media supplemented with 20% fetal bovine sera was seeded on a 12-well plate (In Vitro Scientific, USA). The AlexaFluor 488-modified oligonucleotide (0.25 µl, 1 mM solution, see Preparation of DNA) was incubated with a 12.5 µl FND-PEI complex (1 mg per ml of FND-PEI, sonicated for 30 minutes) for 60 minutes at room temperature. The FND-PEI-DNA complex was isolated by centrifugation at 14 000g for 15 min, and the pellet was resuspended in an appropriate amount of DNAse-free water (Qiagen) to obtain a stock solution of FND-PEI-DNA (1 mg ml -1 ). IC-21 cells were incubated with the FND-PEI-DNA complex (final concentration 25 µg ml -1 ) for 30, 60, and 120 min. In parallel cell samples, an equal amount of the oligonucleotide was delivered into the cells using the commercial transfection reagent X-tremeGENE HP DNA (Roche; 3 : 1 ratio; the manufacturers protocol has been followed). At the end of the incubation period, cells were harvested, washed, and resuspended in PBS. Before the flow cytometry analysis, dead cells were stained with Hoechst 33258 (staining dead cells) for 5 minutes and the samples were measured with a flow cytometer BD LSR II and analyzed with a FlowJo 7.2.2 software.