**3.7 Uptake studies and FACS analysis**

Two days before the uptake experiment 10 000 HAEC and HASMC respectively were seeded onto a 24-well tissue culture plate. One hour before the experiment the culture medium was refreshed with 400 μL of cell specific medium containing 5 % FCS. Prior to the inhibition experiments the cells were incubated with the inhibitors (cytochalasin D (Cch-D, 10 µM) for 120 min, chlorpromazine (chlp, 56 μM), LY29004 (ly, 50 μM), wortmannin (wm, 50 nM) genistein (gen, 200 μM) and nocodazole (noco, 10 μM) for 60 min, filipin (fil, 5 μg/ml for HASMC, 10 µg/mL for HAEC) for 30 min and methyl-β-cyclodextrin (mbCD, 164 μM) for 15 min). Subsequently, the DC-30 based lipoplexes containing Cy5-labeled plasmid DNA were added and the cells were incubated at 37 °C for another 60 min. Then, cells were washed with PBS and detached with 200 µL trypsin/EDTA (0,5 mg/mL trypsin, 0,2 mg/mL EDTA) for 30 s (HAEC) or 90 s (HASMC) and harvested by centrifugation. The cell pellet was washed in one ml ice-cold PBS and resuspended again in 200 µL ice-cold PBS. Cell toxicity was measured by adding 2 µL 7-AAD (BD Biosciences), a fluorescent DNAbinding dye which only penetrates dead cells. Fluorescent positive cells were analyzed with a FACS Calibur using Cell Quest Pro software (BD Biosciences). 10 000 cells were measured for each sample. GFP was measured using fluorescence channel 1 (530+/-15 nm), Rh in fluorescence channel 2 (585+/-21 nm), 7-AAD in fluorescence channel 3 (661+/-8 nm) and Cy5 in fluorescence channel 4 (> 670 nm).

In order to determine the amount of lipoplex absorbed to the outside of the cell membrane, experiments were also performed at 4 °C. Furthermore, control experiments such as incubation only with Cy5-labeled DNA with buffer alone were performed.
