After 48 hours, fresh medium free from NCS was added. Forty-eight hours after this time-point CM was collected, centrifuged at 20 000 g for 3 minutes and the supernatant stored at -80°C as SVF CM. Human PC-3 and LNCaP cell lines PC-3 and LNCaP cell lines were obtained from the European Collection of Cell Cultures (ECCAC) and from the American Type Cell Culture (ATCC), respectively. Both cell lines were maintained in RPMI 1640 medium, supplemented with (%) L-glutamine and (%) Hepes (Gibco), 10% FBS (Gibco) and 1% PS (Sigma Aldrich), at 37°C with 5% CO2. Cell proliferation Cancer cells were seeded into 96-well Enzalutamide manufacturer plates (5×103 and 10×103 cells/well

for PC-3 and LNCaP cells, respectively) and incubated for 24 hours in RPMI 1640 medium with 10% FBS. Next, supernatant AMG510 was removed and new cell medium free from FBS, with (50% volume) or without (control) adipose tissue-derived conditioned medium was added to cancer cells. Media was removed after 24 hours, and cells were stored at -80°C. Then, the pellet was solubilized in a lysis buffer supplemented with a DNA-binding selleck chemicals dye (CyQUANT cell proliferation assay, Invitrogen). DNA content was evaluated in each well by fluorimetry at 480/535 nm using a standard curve previously

generated for each cell type, after plotting measured fluorescence values in samples vs cell number, as determined from cell suspensions using a hemocytometer. Samples were performed in duplicate and the mean value used for analyses. Zymography Gelatinolytic activities of MMP2 and MMP9 of supernatants from adipose tissue primary cultures were determined on substrate impregnated gels. Briefly, Interleukin-2 receptor total protein from supernatants

of primary cultures of adipose tissue (12 μg/well), were separated on 10% SDS-PAGE gels containing 0.1% gelatin (Sigma-Aldrich). After electrophoresis a 30 minutes washing step (2% Triton X-100) was performed, and gels were incubated 16-18 h at 37°C in substrate buffer (50 mM Tris-HCl, pH7.5, 10 mM CaCl2), to allow MMP reactivation. Next, gels were stained in a solution with Comassie Brilliant Blue R-250 (Sigma-Aldrich), 40% methanol and 10% acetic acid for 30 minutes. The correspondent MMP2 and MMP9 clear lysed bands were identified based on their molecular weight and measured with a densitometer (Quantity One, BioRad). Cell tracking and analysis of cellular motility For the time-lapse microscopy analysis (Zeiss Axiovert inverted-fluorescence microscope), exponentially growing cancer cells were seeded into 96-well plates at a density of 5×103 and 10×103 cells/well, for PC-3 and LNCaP, respectively. After 24 hours incubation in RPMI 1640 media supplemented with 10% FBS, supernatant was removed and new medium with (50% volume) or without (control, 0% CM) adipose tissue-derived conditioned medium, were added to cancer cells. At this time point the time-lapse experiment was started.