Eukaryotic cell culture, transfections and creation of stable cell lines

Eukaryotic cell lines used in this study were explained in section 2.1.10.2. All cell lines were cultured in humidified incubators supplied with 5% CO2 at 37°C. Cells were passaged every 3-7 days to a new culture dish and plated 1:5-1:20 dilutions.

HEK293, N1E-115, U343 and COS-7 cells were culture in DMEM 4.5g/l glucose supplemented with 10% heat inactivated FCS and antibiotics (100 units/ml penicillin and 100µg/ml streptomycin). Same medium with 10% horse serum and 5% FCS was used for culturing of PC12 cells

2.2.17.2. Transfection of plasmid vectors and siRNAs

Cells were transfected with plasmid vectors using lipofectamine 2000 reagent when they were 80-90% confluent and still in growth phase. Cells were transfected with DNA/lipofectamine complexes (prepared according to the manufacturers instructions)

in serum and antibiotic free medium (generally Optimem was used) for 3-4 h. At the end of this period, cells were either directly seeded on coated cover-glasses or grown overnight in full medium with serum. Sometimes confluent cells growing on cover-glasses were transfected. In this case, less transfection reagent was used to decrease toxicity and the cells were transfected overnight in full medium.

siRNA transfections were carried out twice using Oligofectamine or Lipofectamine 2000 reagents (Gibco). Confluent cells growing in 6-well plates were transfected with 3µl of 100µM siRNA duplexes complexed with 3µl transfection reagent in serum free medium as before. Cells were replated at 1:5 dilution and grown 48 hours before the next transfection in the same way. 72 h after the first transfection cells were seeded on cover-glasses and analyzed next day. In case of double transfections with plasmid vectors and siRNAs, always lipofectamine 2000 reagent was used during the first transfection.

2.2.17.3. Creation of stable cell lines

HEK293 cells stably expressing pcFLAG-synaptotagmin1, stonin 1, pcHA-stonin 2 and pEGFP-pcHA-stonin 1 were created. All of these vectors contain a neomycin cassette that confers resistance to geneticin, analogous to ES cell selection procedure.

Cells in 6 well plates were transfected with 1µg of the respective constructs. Next day cells were transferred to a 10 cm dish and cultured in presence of 1000µg/ml geneticin (Gibco) for 7-8 days until they reached confluence. All of the control cells which were not transfected had died at the end of this period. Cells were seeded again at 1:10 dilution and cultured in selective medium until they reached confluence.

For selection of single clones, cells were dissociated by trypsinization and cell density was counted. Cells were diluted and resuspended in such a way that cell density would be 1 cell per 100µl. 100µl of the resuspension was seeded into the wells of 96-well plate and cells were cultured in selective medium for 10-20 days until they became confluent. 12-24 putative clones were chosen and transferred to 24-well plates for expanding. Expression levels of transgenic constructs were tested by immunofluorescence microscopy. Clones having mild expression levels in all cells were selected and expanded. These clones were stored frozen in liquid N2.

HEK293 stable cell line expressing lumenal flag tagged synaptotagmin 1 was obtained by expansion of clone 2 and it was called as HEK293-FLAG-Syt1. These cells had roughly equal levels of synaptotagmin 1 stranded on their plasmalemma until passage 10 of the frozen stocks in non-selective medium. After 10 passages some cells ceased to express synaptotagmin1. Whether this can be prevented or delayed by culturing in selective medium has not been tested.

2.2.18. Antibody internalization assays and quantification

HEK293-FLAG-syt1 cells were incubated on ice with 1:500 dilution (roughly10µg/ml) of α-FLAG antibody in Optimem for 30 min for surface labeling. Cells were warmed up to 20°C or 37°C and incubated for the indicated time periods to allow for endocytotic internalization of ligands. Cells were washed twice with PBS and fixed with 4% PFA in PBS for 30 min at RT. Primary antibodies bound to non-internalized surface-stranded synaptotagmin 1 were blocked by incubating the cells with goat-anti-mouse IgG (1:5 dilution) for 2 h at RT in GSDB without triton. Cells were washed once with PBS and permeabilized in regular GSDB and stained with primary and secondary antibodies (diluted in GSDB) as explained before. Cell nuclei were stained by DAPI which was included in the mounting reagent.

For quantification of antibody internalization levels, 3 independent experiments were carried out for each experimental condition. Slidebook 4.0.10 software (Intelligent Imaging Innovations Inc.) was utilized for quantifying the results. Using equal exposure times, 3 fluorescence images were acquired from each experiment giving 9 data sets in total. Each data set contained 10-30 cells (transfected or untransfected depending on the experiment). The fluorescence intensity of the internalized antibody was calculated by subtracting the background fluorescence from the total fluorescence intensity. For experiments using different stonin 2 constructs, the fluorescence intensity was measured only from the transfected cells. The fluorescence intensity of any particular data set represents the average intensity of the cells in that data set. Error bars in the figures depict the standard deviation of values obtained from 9 data sets.

2.2.19. Mouse colony management

Stonin 2 mouse colony was maintained in conventional accomodations. They were kept in rooms that were continuously air conditioned and monitored for temperature and humidity. 12 h light/dark cycles were applied to imitate day and night.

For the initial establishment of the colony, chimeric male mice obtained from microinjection of F11 and A15 ES cell clones were crossed with WT BL6 females.

The agouti colored offsprings were genotyped when they reached 3 weeks age.

Heterozygous animals were crossed between each other to generate the stonin 2 knock-out animals. Knock-out and WT animals were crossed with each other (only identical genotypes were crossed) and animals were maintained in a mixed background (50% 129/ola and 50% BL6). Whenever necessary, genotyping of animals was performed after they reached 3 weeks age (Figure 2.12). For neuronal preparations, neonatal pups were genotyped on the day of birth. Offsprings were separated from their mothers 3-4 weeks after birth.

Genotyping was carried out by PCR using two primer pairs testing for the presence of either neomycin gene(Neo_Fwd1 and Neo_Rev1) or a WT exonic fragment deleted from the stonin2 knock-out loci (Pr-22 and Pr-23). 10-50ng of genomic DNA was amplified in the following PCR reaction using Taq DNA polymerase:

Genotyping PCR

95°C 10 min Initial denaturation 95°C 30 sec Denaturation 60°C 30 sec Annealing 72°C 30 sec Extension 72°C 2 min Final extension

4°C ∞ Storage

Figure 2.12 Genotyping of 3 week-old pups by PCR. Tail DNA from 3 weeks old pups of heterozygous male and female parents was used in genotyping. KO pups are positive only for the neomycin fragment amplified by neomycin primers whereas the WT pups only for WT fragment amplified by P-22/P-23 primers. Control DNA samples were also amplified during genotyping.

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