Cell culture

For the discrete cell lines, following culture media were used:

- MDCK-II, COS-1 cells : DMEM with 1 g glucose / l - Huh7, HT29 cells : DMEM with 4.5 g glucose / l - CHO cells : RPMI with 5% fetal calf serum - CHO/Lec cells : MEM alpha modification

If not indicated otherwise, medium was supplemented with 10% fetal calf serum (FCS), 500,000 U / l penicillin, 10 mg / l streptomycin, 292 mg / l L-glutamine and 110 mg / l sodium-pyruvate.

3.2.3.2 Cell culture procedures

The cell were maintained in coated culture dishes at different sizes (100 mm, 60 mm, 30 mm etc) at 37°C, 95 % relative humidity in an 5 % carbon dioxide atmosphere.

Cells were passaged at 90 - 100 % optical density. Therefore, the cells were washed with PBS and carefully trypsinized with 2 ml trypsin for 15 minutes (in case of HT 29 and Huh7 cells a longer time period was necessary). Subsequently, trypsin activity was stopped with culture medium, and the cells were collected in suspension. After centrifugation and decanting of the supernatant fresh culture medium was added and the cells were spread in new culture dishes at the desired density (mostly 5 x 10³ cells / cm²) which was calculated by counting suspended cells in a Thoma chamber.

The culture medium was replaced every day or every two days, depending on cell type and density.

Long time storage of mammalian cells was performed in liquid nitrogen at -196°C.

Thereby, cells were trypsinized and collected as described above. Instead of fresh culture medium, fetal calf serum, supplemented with 10 % dimethyle sulfoxide (DMSO) was used to freeze the cells in kryoconservation tubes first at -80°C. On the next day, tubes were transferred into liquid nitrogen.

After thawing of cell aliquots, the DMSO-containing serum was immediately removed and replaced by fresh culture medium in order to prevent the cells from the toxic effects of DMSO.

3.2.3.3 Transient transfection

COS-1 cells were transfected using the diethyle-amino-ethyle (DEAE)-dextrane method (modified according to Naim et al., 1991). This technique is based on the negative polarity of plasmid DNA, which forms stable complexes with positive charged DEAE-dextrane molecules. Incubated with eukaryotic cells, these complexes attach to the negative charged cell surface and are occasionally phagocytosed. Therefore, cells were split the day before on 100 mm dishes transfection in order to achieve a cell density of 30 – 40 %.

For formation of stable dextrane-DNA complexes, 5 µg plasmid DNA was dissolved in 1.5 ml culture medium which did not contain FCS. After 5 minutes, 10 µl of stock solution DEAE-dextrane (50 mg / ml H2O) were added to obtain a final concentration of 0.5 mg DEAE-dextrane / ml culture medium. The solution was mixed thoroughly and incubated for approximately 30 minutes in order to form stable DNA-dextrane complexes. The cells were washed with PBS and the DNA-dextrane mix was dispersed onto the cells. Within the next 90 minutes, tissue plates were shaked every 15 minutes to assure proper covering of all cells. During this period, plasmid DNA is incorporated by the cells.

Afterwards, the transfection mix was discarded and the cells were washed twice with PBS. In order to enhance the transfection rate, the cultures were subsequent incubated with cloroquine at 50 µg / ml final concentration. This reagent prevents the degradation of DNA by blocking the fusion of plasmid containing phagosomes with the lysosomes. For confocal analysis, the enhancer was removed after 1 hour, for biochemical experiments after 4 hours. Cells were finally washed and culture medium was changed the day after transfection again. Mostly, cells were processed 48 after transfection, because then protein expression reaches maximal levels.

MDCK-II, CHO, HT29 and Huh7 cells were transfected with Lipofectamine^TM 2000.

Similar to the described transfection with DEAE-dextran, the positive charged cationic lipid reagents forms complexes with plasmid DNA. According to the manufacturer’s protocol, for 60 mm dishes 10 µl transfection reagent and 4 µg of plasmid DNA dissolved in 1 ml culture medium without FCS and without antibiotics were used. After 20 minutes incubation, cells were washed with PBS and the transfection mix was added to the cells for 6 – 8 hours. Due to the lipohilic character of the reagent, the plasmid-cation complexes merge in the plasma membrane and finally the plasmid DNA is incorporated into the cytoplasma.

3.2.3.4 Establishment of stable cell lines

For establishment of stable cell lines, CHO and MDCK-II cells were were transfected with Lipofectamine^TM 2000 as described above.

The day after transfection, cells were trypsinized, carefully singularized and split 1 : 30 onto new plates. After one more day the culture medium was changed and 500 µg / ml Geneticin^®. Every four days, medium was replaced and fresh antibiotic was applied. Thereby, only cells which have stably incorporated the plasmid into their genome, coding for a Geneticin^®-resistance, are able to inactivate the antibiotic and survive. Two weeks after transfection positive clones were identified by confocal laser microscopy and selected for culturing. From each construct, at least three monoclonal cultures were established and the transfection rate and correct intracellular protein processing was estimated by confocal laser microscopy and western blot. An overview of constructed stable cell lines is given in Table 2.

Identification Cell line Transfected construct Transfection efficiency MDCK-PLKC-YFP MDCK-II pEYFP-PLKC ~ 99 % MDCK-PLKC∆PDZ-YFP MDCK-II pEYFP-PLKC∆PDZ ~ 99 % MDCK-PLKC∆Cyt-YFP MDCK-II pEYFP-PLKC∆Cyt > 80 % MDCK-PLKC∆TM-YFP MDCK-II pEYFP-PLKC∆TM > 60 % MDCK-PLKC∆6-YFP MDCK-II pEYFP-PLKC∆6 > 90 %

MDCK-PLKC-myc MDCK-II pEYFP-PLKC ~ 40%

CHO-PLKC-YFP CHO pEYFP-PLKC > 95 %

CHO-PLKC∆Cyt-YFP CHO pEYFP-PLKC∆Cyt ~ 40%

CHO-PLKC∆6-YFP CHO pEYFP-PLKC∆6 ~ 99 %

CHO-E-cadherin-GFP CHO pEGFP-E-cadherin ~ 99%

CHO-N-cadherin-GFP CHO pEGFP-N-cadherin ~ 99%

CHO/Lec1-PLKC-YFP CHO/Lec1 pEYFP-PLKC 100 % CHO/Lec2-PLKC-YFP CHO/Lec2 pEYFP-PLKC 100 % CHO/Lec8-PLKC-YFP CHO/Lec8 pEYFP-PLKC 100 %

Table 2 Stably tranfected cell lines

3.2.4 Isolation and detection of proteins