PRESENTACION E INTERPRETACION DE RESULTADOS Cuadro Nº 10

2.5.1 Invasion Assay

Invasion assays were performed using the method of Albini (1998). ECM (Sigma E-1270) (llm g/lm l) was diluted to lmg/ml in serum-free DMEM. lOOfil o f lmg/ml ECM was placed into each invasion chamber/insert (Falcon 3097) (8.0(j,m pore size, 24 well format) which was placed in a 24-well plate (Costar). This was carried out on ice to prevent the ECM from solidifying during the process. The inserts were incubated overnight at 4°C. The following day, the cells were harvested and re-suspended in media-containing fetal calf serum, at a concentration of lxlO6 cell/ml. The inserts were washed with serum-free DMEM, then lOOpl of the cell suspension were added to each insert and 300jil o f media containing serum was added to the well underneath the insert. Cells were incubated at 37°C for 48 hours. After this time, the inner side o f the insert was wiped with a wet cotton swab while the outer side of the insert membrane was stained with 0.25% crystal violet for 10 minutes and then rinsed with PBS and allowed to dry. The inserts were then viewed under the microscope

The procedure for the ready-coated inserts was very similar to the procedure described above, with the exception that the inserts were not coated with ECM (as they were pre­ coated with matrigel), but were re-hydrated for 2 hours prior to use, by adding serum-free media and incubating at 37°C, following the manufacture’s instructions (Beckton Dickinson).

For a quantitative method of invasion analysis, assessment o f cell migration to the outer side of the insert was determined by acid phosphatase assay (Section 2.3.1.2). Cells were incubated for the time indicated above, after which, the inner side o f the insert was wiped with a wet cotton swab and the media in the well was replaced with 200ul of freshly prepared phosphatase substrate (lOmM /»-nitrophenol phosphate (Sigma 104-0) in 0.1M sodium acetate (Sigma, S8625), 0.1% triton X-100 (BDH, 30632), pH 5.5). The plates were wrapped in aluminium foil and incubated in the dark at 37°C, 5% CO2, for 2 hours.

The enzymatic reaction was stopped by the addition of 50 (j.1 o f 1M NaOH to each well. lOOfol aliquots were removed from the wells, placed in a 96-well plate, and read in a dual beam plate reader at 405 nm with a reference wavelength o f 620 nm,

Another quantitative method was also used in come cases whereby the crystal violet dye was eluted with 200 (0.1 of 33 % glacial acetic acid/well. 100 |J,1 aliquots were transferred from each well to corresponding wells of a 96 well plate and the absorbance was read at 570 nm.

2.5.1.1 Invasion Inhibition Assays

The procedure for carrying out MMP inhibition assays was identical to that of invasion assays (section 2.5.1), with the exception that 200(j.g/ml MMP inhibitor I, 10(j.g/ml MMP inhibitor HI or 10 jog/ml MMP-2 inhibitor I was added both to the cell suspension in the insert and to the medium in the well underneath the insert before the cells were incubated at 37°C, 5%C02, for 48 hours. All three MMP inhibitors were purchased from CalBiochem (Nottingham, UK). Table 2.5.1 illustrates details provided by manufactures about the function of each inhibitor

Table 2.5.1 MMP inhibitors used in invasion inhibition assays and the MMP whose activity they inhibit.

Inhibitor MMP inhibited Catalogue number

MMP inhibitor I MMP-1, -3, -8 and-9 444250 MMP inhibitor IE MMP-1, -2, -3, -7 and-13 444264

2.5.2 M otility A ssay

The procedure for carrying out motility assays was identical to the procedure used for invasion assays (section 2.5.1) with the exception that the inserts were not coated with ECM. Incubation times were 12, 24 and 48 hours. The procedure for quantitative analysis was identical to that of the invasion assays.

2.5.3 Adhesion Assay

Adhesion assay was preformed using the method o f Torimura et al. (1999). Collagen Type IV (Sigma C-5533), fibronectin (Sigma F-2006) and laminin (Sigma L-2020) were reconstituted in PBS to a stock concentration o f 500(j,g/ml. Stocks were aliquoted into sterile Eppendorfs. Fibronectin and collagen stocks were stored at -20°C and laminin stocks were stored at -80°C. ECM (Sigma E-1270) was diluted to lmg/ml in serum-free DMEM, aliquoted and stored at -20°C. ECM undergoes thermally activated polymerization when brought to 20-40°C to form a reconstituted basement membrane.

When the solutions were ready for use, each of the ECM proteins, collagen, fibronectin and laminin, was diluted to 25jog/ml with PBS, while ECM was diluted to lmg/ml with serum- free DMEM. 250(0.1 aliquots were placed into wells of a 24-well plate. The plates were tapped gently to ensure that the base o f each well was completely covered with solution. The plates were then incubated at 4°C overnight. The ECM solutions were then removed from the wells and the wells were rinsed twice with sterile PBS. 0.5ml of a sterile 0.1% BSA/PBS solution was dispensed into each well to reduce non-specific binding. The plates were incubated at 37°C for 20 minutes and then rinsed twice with PBS.

Cells were harvested and resuspended in serum-free DMEM medium. The cells were then plated at a concentration of 2.5x104 cells per well and incubated at 37°C for 60 minutes. After that time, the media was removed from the wells and the wells were rinsed gently with PBS. The cells were then stained with 0.5ml of 0.25% crystal violet dye for 10 minutes. The plates were then rinsed and allowed to dry. The dye was eluted with 200(xl o f

33% glacial acetic acid and lOOjal aliquots were transferred to a 96-well plate and the absorbance was read at 570 nm (Torimura et al., 1999).

2.5.4 Zymography

Zymography was used to assess the level o f proteolytic activity o f different proteinases. The choice o f substrate incorporated into the resolving gel depends on substrate specificity of the species of enzyme to be detected (Johansson et al., 1986). Gelatin is a substrate for matrix metalloprotienases (MMPs), serine and cysteine proteinases.

The gel was prepared by incorporating gelatin within the polymerized acrylamide matrix. 10% acrylamide gels were used. The quantities for one gel is given in Table 2.5.4a.

Table 2.5.4a Preparation of resolving and stacking gel for zymography

Components Resolving gel (10%) Stacking gel

Acrylamide stock* 3.3 mis 0.5 mis

3mg/ml Gelatin 2.5 mis

1.875M-Tris/HCl, pH 8.8 2.5 mis

1.25M-Tris/HCl, pH 6.8 “ 0.8 mis

Ultrapure water 1.7 mis 2 mis

10% Ammonium persulphate (Sigma, A-1433) 33 jils 33 jj!s TEMED (Sigma, T-8133) 5 (ils 5 pis

* Acrylamide stock solution consists o f 2 9 .lg acrylamide (Sigma, A8887) and 0.9g N N ’-methylene bis- acrylamide (Sigma, 7256) dissolved in 60ml UHP water and made up to 100ml final volume. The solution was stored in the dark at 4°C for up to 1 month. A ll components were purchased from Sigma, SDS (L-4509), NH4- persulphate (A-1433), TEMED, N ,N ,N ,N ’-tetramethylethylenediamine (T-8133) and Gelatin (G-8150).

Cells were grown in cell culture Petri dishes (Greiner). When the cells were 80% confluent, they were rinsed twice with sterile PBS, followed by 4-hour incubation with serum-free medium. The cells were then grown in fresh serum-free medium for another 24-72 hours (depending on their growth rate). After the relevant time period, supernatants were collected as samples. Samples were mixed 3:1 with 4X sample buffer (10% glycerol; 0.25M Tris-HCl, pH 6.8; 0.1% (w/v) bromophenol blue) and were loaded onto the gel. 5(4.1 of boiled Broad size range protein marker (New England Biolabs, 7708S) was also loaded onto the gel. The gels were run at 30 mA per gel in running buffer (as in section 2.4.1.3) until the dye front reached the bottom o f the gel. Following electophoresis, the gels were soaked in 2.5% Triton-X-100 with gentle shaking for 30 minutes at room temperature. The gels were then rinsed in substrate buffer (50mM Tris-HCl, pH8.0; 5mM CaCh) and then incubated for 24 hours in substrate buffer at 37°C. The gels were then stained with Coomassie blue (2.5mg/ml) for 2 hours by shaking and destained in destain solution (50ml acetic acid; 150ml isopropanol; 300 ml distilled water) until clear bands were visible. The gels were then scanned.

To identify the different classes o f proteinases that are being secreted, inhibitors of proteinases were added to 2.5% Triton-X-100 in substrate buffer. The inhibitors used in this study are listed in Table 2.6.4b.

Table 2.5.4b Inhibitors of different classes o f proteinases used in gelatin zymography

Inhibitor

Enzyme inhibted

Concentration used

EDTA MMPs 30 mM