Cellular biology methods

Cultivation of eukaryotic cell lines

For all experiments HEK293 cells were used. They were cultured in DMEM (Dul-becco’s modified Eagle’s minimal essential medium) (Thermo Fisher Scientific, Waltham) containing 1 % penicillin/streptomycin and 10 % fetal calf serum (FCS).

The media for stably transfected cell lines also contained 0.4-0.8 g/l G-418 as a selecting agent.

Freezing and thawing of cells

Cells can be frozen and stored in liquid nitrogen and thawed again, when needed.

Whenever possible cells from early passages were used. Usually cells were not used passed passage 20.

Freezing of cells In order to prevent crystal formation and cell death during the freezing process, DMSO was added as a cryoprotective agent. The cells were washed once with PBS (phosphate buffered saline) and then removed with trypsin.

The cells were resuspended in 1 ml per cryotube normal cell medium. One 10 cm plate with confluent cells was sufficient for four cryotubes. The cells were transferred into prechilled cryotubes. To one ml of cells one ml of cold freeze medium was added. The cells were stored for 24 hours at -20^◦C and for another 24 hours at -80^◦C. On the third day the cells were transferred into liquid nitrogen storage.

Freeze medium (2x)

FCS 20 %

DMSO 40 %

normal culture medium 40 % cool freeze medium before use

Thawing of cells The cells were taken out of liquid nitrogen and directly defrosted at 37^◦C. Once the medium was thawed, they were put in a cell culture dish with fresh prewarmed (37^◦C) medium and incubated until the cells had attached. Then the medium was changed in order to remove the cryoprotective DMSO, which is toxic for cells, when they are not frozen.

Transient transfection of HEK293 cells

HEK293 cells were transfected using Effectene (Qiagen, Hilden). Depending on the following experiment either a regular transfection or a fast-forward transfection was performed. The difference between the two approaches is that for a regular transfection the cells are passaged the day prior to the transfection and cultivated to a confluency of 80 %. They undergo a media exchange and the transfection reagent is pipetted onto the adherent cells. In a fast-forward transfection the cells were passaged on the same day as the transfection and the transfection reagent was pipetted onto the cells, while they were attaching. The transfection efficiency was similar between those two approaches.

To condense the expression vector plasmid, the DNA, EC buffer and Enhancer were mixed and incubated for five minutes. In a following step the transfection reagent Effectene was added. It was mixed and incubated for ten minutes, where it formed a complex with the DNA. This transfection-complex was slowly given onto the cells, where it was able to pass the cell and the nuclear membrane, which led to the production of the desired protein.

Diameter 3.5 cm 10 cm 15 cm

DNA 0.4µg 2µg 4µg

EC buffer 100µl 250µl 500µl

Enhancer 3.2µl 16µl 32µl

vortex 5 s, incubate at room temperature 5 minutes

Effectene 10µl 60µl 120µl

vortex 10 s, incubate at room temperature 10 minutes

DMEM 600µl 3 ml 7 ml

The transfection efficiency of the transient transfection was approximately 70 %.

Stable transfection of HEK293 cells

One 10 cm dish of HEK293 cells was transfected as described above. 0.8 g/l of Geneticin (G-418) was added to the normal cell culture medium. Starting on the second day after transfection, the medium was exchanged daily. Only transfected cells, which integrate the G-418 resistance gene into their genome can produce the protein, that is responsible for the antibiotic resistance. These cells survived the selection process, while all untransfected cells and cells that were successfully transfected, but did not integrate the resistance gene into their genome died.

The daily medium exchange was stopped when the cells started to proliferate again. Usually this took 14 days.

Flow cytometry FACS buffer

BSA 1 %

EDTA 1 mM

in PBS

sterile filtered (0.2µm)

Cells were measured and sorted in a Bio-Rad S3 sorter. Depending on the experiment 1.5 x 10⁶ to 4 x 10⁶ cells were used.

The cells were detached by pipetting up and down in PBS and then centrifuged at 300 x g for 5 minutes. The cells were then immediately put on ice and washed once in FACS buffer. The primary antibody was diluted in FACS buffer to a concentration of 5µg/ml in 200 - 400µl and the samples were incubated for one hour at 4^◦C in an end-over-end shaker. The cells were then washed three times in FACS buffer and incubated in the secondary antibody (5µg/ml in 200 - 400µl) for one hour at 4 ^◦C in an end-over-end shaker. After incubation in the secondary antibody the cells were again washed three times and then measured in the Bio-Rad S3 sorter.

When the cells were sorted, all washing steps were carried out in a sterile cell culture hood. The cells were sorted into 500µl of FACS buffer at 4 ^◦C and immediately after the sorting procedure put on ice. Then they were centrifuged at 300 x g for 5 minutes and the resulting pellet was resuspended in warm DMEM medium containing 2 % penicillin/streptomycin and 0.8 g/l G-418 as antibiotics.

SILAC

For SILAC labeling HEK293 cells were cultivated in heavy and light medium, respectively. For labeling the SILAC-Lys6-Arg10-Kit from Silantes (Munich) was used. The cells were grown in SILAC medium for one week and then a part of the cells was lysed in 3 % NP-40 lysis buffer. Full labeling was verified using mass spectrometry. The labeled cells were then frozen and stored in liquid nitrogen.