The average

number of cells/field was then multiplied by a factor of 140 (growth area of membrane/field area viewed at 200× magnification (calibrated using a microscope graticule)). The selleck chemicals llc mean values were obtained from a minimum of three individual experiments and were subjected to t -tests and ANOVA. Motility assays were carried out in the same manner as invasion assays without the addition of ECM on the insert. Experiments were performed in triplicate. Adhesion assay Adhesion assays were performed using a modified method [21]. 24-well plates were coated with 250 μl of 25 μg/ml ECM proteins (laminin, fibronectin and collagen type IV), 10 μg/ml of collagen type I and 1 mg/ml of matrigel. ECM proteins were incubated overnight at 4°C. To reduce non-specific binding, 0.5 ml of 0.1% BSA-PBS solution was added to each well and incubated for 20 minutes, then rinsed twice with sterile PBS. A single cell suspension was obtained, 1 ml of a 2.5 × 104 cell suspension was added onto the pre-coated 24-well plates in triplicate and allowed to attach for 60 minutes. Blank wells learn more contained ECM proteins but no cells; controls were uncoated wells with cells. After 60 minutes, the non-adhered cells were removed

by washing twice with sterile PBS. 200 μl of freshly prepared phosphatase substrate (10 mM p -nitrophenol phosphate in 0.1 M sodium acetate, 0.1% Triton X-100 pH 5.5) was added to each well. Plates were then incubated in the dark at 37°C for 2 hours. The enzymatic reaction was stopped by the addition of 100 μl 1 M NaOH. The absorbance was read on a BIO-TEK plate reader at 405 nm with a reference wavelength of PCI-32765 research buy 620 nm. Anoikis assay 24-well plates selleck were coated with 200 μl of poly-2-hydroxyethyl methacrylate (poly-HEMA, 12 mg/ml dissolved in 95% ethanol, Sigma) and allowed to dry overnight. 1 ml of a single cell suspension of 1 × 105cells was plated onto standard 24 well plates or poly-HEMA coated plates. After 24 hours incubation at

37°C in a humidified atmosphere containing 5% CO2, the viability of cells was quantitatively measured using alamarBlue indicator dye (Serotec). The absorbance was read on a BIO-TEC plate reader at 570 nm with a reference wavelength of 600 nm. Soft agar colony-forming assay Soft agar assays or anchorage independent growth assays were carried out using a modified method [22]. 1.548 g of agar (Bacto Difco, 214040) was dissolved in 100 ml of ultra pure water and autoclaved. This agar was then melted in a microwave oven immediately prior to use and incubated at 44°C. 50 ml of agar was then added to 2× DMEM AgarMedium (AgM), mixed well and quickly dispensed onto 35 mm sterile petri dishes. The plates were allowed to set at room temperature and the remaining AgM was returned to the water bath with the temperature reduced to 41°C. 10% FCS was added to the AgM. Cells were harvested and resuspended in medium without serum, ensuring that a single cell suspension was obtained.