2 MATERIALS AND METHODS
2.2 M ETHODS
2.2.2 Tissue Culture
Stable clones of A2780-‐DNA3 and A2780-‐Rab25 were generated as previously described by Cheng et al. [351] and generously provided by Gordon Mills. MDA-‐MB-‐231 cells were
purchased from the American Type Culture Collection cell bank. Various other cell lines used in this project were obtained from research groups within the Beatson Institute for Cancer Research, including Telomerase-‐immortalised human fibroblasts (TIFs) and colon carcinoma BE cells.
2.2.2.2 Cultivation of cells
A2780 cells were cultured in RPMI-‐1640 medium supplemented with 10% (v/v) foetal calf serum and 2 mM L-‐glutamine at 37°C in a humidified atmosphere containing 5% CO2. All other cell lines were cultured in DMEM containing 10% (v/v) foetal calf serum and 2 mM L-‐glutamine under the same conditions as described above. When cells reached 80% confluence, they were sub-‐cultured. To do this, the culture medium was removed, the cellular monolayer then rinsed with PBS and a 10% Trypsin/PE solution was added.
After a brief incubation, the detached cells were re-‐suspended in the appropriate cell culture medium and centrifuged for 2 minute at 900 rpm. Next, the cell pellet was resuspended in fresh medium and an aliquot was seeded into an appropriate tissue culture dish or flask.
2.2.2.3 Freezing and thawing of cells
At least 1x106 cells in excellent condition were trypsinised and centrifuged for 2 minutes at 900 rpm. Once the supernatant was completely removed, the cell pellet was
resuspended in 1 ml Cryo-‐SFM medium and transferred into a labelled cryo vial and placed in a freezing container (NALGENE™ 1°C Cryo Freezing Container) at -‐80°C. When cells were to be stored longer than 6 months they were transferred into liquid nitrogen the following day.
According to the rule of “freeze slowly, thaw fast”, frozen cells were put immediately into a 37°C water bath. When only a small lump of ice was left, the vial was opened and the cells were immediately transferred into 5 ml of pre-‐warmed medium. After centrifugation for 2 minutes at 900 rpm, the supernatant was removed and cells were resuspended in their specified growth medium and plated onto a cell culture dish. The following morning, the medium was replaced.
2.2.2.4 Transfection using the Amaxa™ Nucleofector™
Amaxa nucleofection provides an efficient way to introduce DNA plasmids or siRNA oligos. Hence, this method was used for A2780-‐Rab25 and MDA-‐MB-‐231 cells, which were grown to 70-‐85% confluency, trypsinised and resuspended in growth medium.
Depending on the cell line between 1-‐5x106 cells were used for one transfection reaction.
The appropriate number of cells were centrifuged at 900 rpm for 2 min. The supernatant was aspirated off and 100-‐200 pmol of siRNA or appropriate amounts of DNA were added on top of the cell pellet. Afterwards, 100 µl of solution T (A2780-‐Rab25) or
V (MDA-‐MB-‐231) were added and mixed thoroughly by pipetting up and down. The mixture was then transferred into a Nucleofector cuvette and inserted into the AmaxaTM NucleofectorTM. Transfections were carried out using the appropriate programme (see table below). Subsequently, transfected cells were taken up in 500 µl pre-‐warmed growth medium and seeded onto tissue culture dishes. Transfected cells were allowed to recover and settle for at least 12 hours prior to any experiments.
Cell line Nucleofection programme
A2780 A-‐023
MDA-‐MB-‐231 X-‐013
Typically, 3 µg of DNA plasmid were transfected unless otherwise stated in the table below:
Plasmid Transfected amount (µg)
GFP 1
Destination vector 1
SF-‐ERK1 0.4
SF-‐ERK2 1.5
2.2.2.5 Transfection using HiPerFect
For RNA interference experiments, MDA-‐MB-‐231 and BE cells were transfected using HiPerFect, because this increased cellular viability. When cells reached 80% confluence, a
solution of 500 µl of DMEM supplemented with L-‐glutamine only, 7.5 µl HiPerFect and 1.5 µl siRNA (from 20 µM stock) was prepared and left at room temperature for 10 minutes to allow transfection complex formation. Next, the mixture was added onto the cells and the plates were gently swirled to ensure a uniform distribution. Transfected cells were then incubated under normal growth conditions. Two days later, the cells were passaged at a ratio of one in three and transfected immediately after sub-‐culturing following the above protocol. After the second round of transfection gene silencing was monitored by Western blotting or qRT-‐PCR.
2.2.2.6 Cell proliferation assays
10.000 cells were seeded per 6-‐well dish and left to settle overnight. The following morning cells were counted for the first time. For each measurement, cells were
thoroughly trypsinised in 200 µl of Trypsin/PE solution. Next, the trypsin was inactivated with 200 µl of medium. The cell number was determined by adding 20 ml of PBS to the 0.4 ml of cell suspension and measuring the cell number with a Casy® 1 cell counter. Cell proliferation was assayed over the course of 5-‐6 days. Relative cell numbers were
determined by using day one as a reference point. Every condition was set up in triplicate and the experiments were repeated twice. Moreover, Casy® 1 cell counter measurements were checked manually using a haemocytometer.
2.2.2.7 Inverted invasion assay
Inverted invasion assays, previously described by Hennigan et al. were modified as follows [352]. An aliquot of Matrigel was slowly thawed on ice and then diluted 1:1 in ice-‐cold PBS supplemented with 25 µg/ml fibronectin. Subsequently, 100 µl of this solution was carefully pipetted into a Transwell (pore size of 8 µm), which had been inserted into a well of a 24-‐well tissue culture plate. In order for the Matrigel to polymerize, the plate was incubated for at least 30 minutes at 37˚C. Meanwhile, cell suspensions between 1x105 and 4x105 cells per ml were prepared in the appropriate growth medium. After the Matrigel set, the Transwells were inverted and 100 µl of the cell suspension were pipetted onto the underside of the filter. The Transwells were then carefully covered with the base of the 24-‐well tissue culture plate so that it contacted the droplet of cell suspension. Next, the inverted plate was incubated at 37˚C, 5% CO2 for 4 hours to allow cells to attach to the filter. Afterwards, the Transwells were turned
right-‐side-‐up, washed by sequential dipping in 1 ml of the appropriate serum-‐free growth medium twice and finally placed into 1 ml of the serum-‐free medium. Then, 100 µl of growth medium containing 10% serum and the chemoattractant EGF (25ng/ml) was gently pipetted on top of the Matrigel. Cells were allowed to invade into the Matrigel over a course of 2-‐3 days at 37˚C and 5% CO2.
In order to visualize the migrated cells, the Transwells were transferred into a new 24-‐well tissue plate and covered drop-‐wise with 1 ml of calcein acetomethyl ester diluted to 4 µM in serum free medium. After one hour at 37˚C samples were assayed using a LEICA SP2 confocal microscope with a 20x objective. The fluorescent dye was excited with a wavelength of 488 nm and emitted at 515 nm. Optical sections were captured at 15 µm intervals starting from the underside of the Transwell filter and moving upwards in the direction of cell invasion. The resulting images were quantified using the area calculator tool from the ImageJ software. The threshold fluorescence intensity was set so that background intensities were erased and only cells within the optical slice were visualised.
The sum of fluorescence of the sections from 45 µm and above was divided by the total fluorescence of all the optical sections, thus giving an invasion index of ≥45 µm. Mean values were generated from three individual experiments. Within one experiment each condition was set up in duplicate and optical sections for three areas were taken for each Transwell.
2.2.2.8 Generation of cell-derived matrix
Cell-‐derived matrix was generated as previously described [38, 353]. Briefly, tissue culture plates were coated with 0.2% sterile gelatine for one hour at 37˚C. Next, the gelatine solution was aspirated off and plates were washed twice with PBS. In order to crosslink the layer of gelatine, 1% sterile glutaraldehyde was added for 30 minutes at room temperature. Following two washes with PBS, the crosslinking reaction was quenched with 1 M sterile glycine in PBS (pH ~7) for 20 minutes at room temperature. After another 2 washes, the tissue culture dishes were equilibrated with DMEM containing 10% foetal calf serum and 2 mM L-‐glutamine. Then, human telomerase-‐immortalised fibroblasts were plated at near confluence (~2x104/cm2). The following day, collagen production was stimulated through supplementation of the medium with 50 µg/ml ascorbic acid.
Fibroblasts were cultured for 10-‐14 days and the medium (supplemented with ascorbic
acid) was exchanged every other day. Matrices were then denuded of living fibroblasts by incubating the dishes with PBS containing 20 mM NH4OH and 0.5% Triton X-‐100 for 2 minutes, after which cell lysis was examined using a phase light microscope. Next, the extraction buffer was aspirated off and matrices were washed twice in PHS containing calcium and magnesium. Residual DNA was digested with PBS containing calcium, magnesium and 10 µg/ml DNase1 at 37˚C for 30 minutes. Following another two washes with PBS containing magnesium and calcium, matrices were stored at 4˚C in the same solution supplemented with pen/strep and fungizone. Before use, the matrix condition was examined by phase light microscopy. Prior to seeding the cell line of interest, matrices were washed twice with PBS and equilibrated with complete medium for 20 minutes at 37˚C.
2.2.2.9 Migration on cell-derived matrix
Respective cells (50,000 cells for A2780-‐Rab25 cells, 100,000 cells in the case of MDA-‐MB-‐231 cells) were seeded into a well of a 6-‐well dish coated with cell-‐derived matrix and allowed to adhere for 4 hours prior to imaging. Migration was monitored using a Nikon time-‐lapse microscope and migration characteristics were analysed using the Manual Tracking and Chemotaxis tool of ImageJ and a customised MATLAB script written by Dr. Marc Birtwistle.
2.2.2.10 Scratch wound assays
Cells were seeded into a 6-‐well dish, so that they reached confluence 48 hours post transfection. Next, a wound was introduced by scratching the plastic dish with a 200 µl pipette tip. Following three washes in fully-‐supplemented medium wound closure was monitored at 10 minute intervals using a time-‐lapse microscope. Images for each condition were taken from five distinct fields along the wound and a minimum of five cells were tracked for each field. Migration characteristics from three independent experiments were analysed using the ImageJ cell tracking software.
2.2.2.11 Immunofluorescence
Cells were cultured on sterile coverslips placed in a 6-‐well tissue culture dish. Where indicated, coverslips were coated with fibronectin (20 µg/ml in PBS) for 20 minutes at 37˚C. Medium was removed and the cells were washed twice with PBS and fixed in a 4%
paraformaldehyde solution. After 15 minutes, the crosslinking reaction was quenched by adding an equal volume of a 2M glycine solution. Next, the coverslips were washed twice with PBS, after which cells were permeabilised with 0.05% Triton X-‐100 in PBS for
2 minutes, which was followed by 3 washes in PBS. In order to block non-‐specific antibody binding, cells were incubated in PBS containing 1% BSA for one hour. Then, primary antibodies diluted in blocking solution were applied for one hour. Following another three washes, secondary antibodies in blocking solution were applied for 45 minutes.
Coverslips were washed another two times with PBS before being mounted onto glass slides using Vectashield containing DAPI. Slides were dried overnight at 4˚C. Colocalisation studies were performed on a Zeiss 710 upright confocal microscope with a 64x objective.
Different fluorescent channels were recorded sequentially to prevent bleed-‐through.
2.2.3 Protein biology