**3.8 Spectral bio-imaging**

80 Non-Viral Gene Therapy

lipoplex samples. Analysis was carried out by means of flow cytometry (see uptake and

Cells were cultivated to 80 % confluency as described above. Every 100 mm plate was trypsinated with 1 mL Trypsin/EDTA for 30 s (HAEC), and 90 s (HASMC) to detach the cells. Trypsinization was stopped by adding 3 ml culture medium with serum. After washing twice with ice-cold PBS, cells were redispersed in ice-cold electroporation buffer (100 mM Hepes, 137 mM NaCl, 4 mM Na2HPO4, 6 mM Dextrose) at a concentration of 107 cells/ ml. Electroporation experiments were carried out using a Gene Pulser II (BioRad Laboratories, Muenchen, Germany). 500 µL of cell suspension was poured in a chilled 0,4 cm gap cuvette. After adding 20 µg DNA (1 mg/mL TE-buffer pH 7,0; Tris 10 mM, EDTA 1 mM) the cuvette was placed on ice and electroporated with the following settings: for HAEC: 350 V, 750 µF; for HASMC: 500 V, 950 µF. After the shock, the cuvette stayed on ice for 10 min. Then, cells were transferred into a 100 mm culture plate, (gelatine-coated plates for HAEC, see section "cell culture") and incubated for 48 h at 37 °C and 5 % CO2.

Fluorescent pictures were taken with an Axiovert S 20 (Zeiss, Jena Germany, 20x).

As a positive control experiment, C3-Toxin plasmid was electroporated into the cells to see if DNA was delivered into the nucleus. Effective delivery was achieved when cells were

As cells were not vital enough for FACS analysis, fluorescent and non-fluorescent cells were counted via "Neubauer Zählkammer". Counting was repeated twice for every culture plate

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

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.

FACS analysis).

rounded up and dead.

for at least 3 independent experiments.

**3.7 Uptake studies and FACS analysis** 

Cy5 in fluorescence channel 4 (> 670 nm).

**3.6 Electroporation experiments** 

Cells were seeded onto 12-mm coverslips in 24-well plates 2 days prior to the experiment. Lipoplexes were prepared using DC-30® and Rh-labeled plasmid. Cells were then washed twice with PBS w/o calcium and magnesium and then incubated with 400 µL medium containing the lipoplexes. Cells were incubated simultaneously with the markers for the different pathways for 30 min to 2 h (tf alexa fluor 488 (10 µg/mL), chltx-B alexa fluor 488 (2 µg/mL) and FITC dextran 70 000, (1 µg/mL)). The amount of DNA was the same as that used in the uptake experiments. Prior to microscopic examination, the cells were fixed with 4 % paraformaldehyde (500 µL, 10 min, RT) and the coverslips were mounted on glass slides with 3 µL MobiGlow (MoBiTec, Goettingen, Germany), an antifading substance to reduce photobleaching effects.

Spectral bio-imaging was performed as described previously by Huth et al. [8] with a SpectraCube SD-200H system (Applied Spectral Imaging, Migdal HaEmek, Israel). An inverted fluorescence microscope (Axiovert S 100, Zeiss, Jena Germany) equipped with a high-pressure mercury lamp (HBO 100) for excitation and a triple bandpass filter set was used. All images were taken using a 100x/1.3 oil-immersion objective lens (Plan Neofluar, Zeiss, Jena, Germany). In the spectral range from 400 to 700 nm the objective lens shows only minimal fluctuations of transmission (85-90 %). The optical head attached to the microscope is composed of a Sagnac common-path interferometer and imaging optics including a cooled CCD camera (Hamamatsu C4880-85, Japan). Microscopic images were obtained with spectral bio-imaging 2.5 software (Applied Spectral Imaging). The acquisition time of a desired image varied from 30 to 90 s depending on the brightness of the fluorescence and the image size. Cells were first incubated with only one dye to get singlecolored images. For further analysis images were then transferred to the SpectraView 1.6 software (Applied Spectral Imaging).
