1 to 100 μg/ml) of the tested agents. The compounds were dissolved in 10% DMSO to concentration of 1 mg/ml, and subsequently diluted in culture medium to reach the required

concentrations. DMSO, which was used as a solvent did not exert any inhibitory effect on cell proliferation. The cells attached to the plastic were fixed by gently layering cold 50% TCA (trichloroacetic acid, Aldrich-Chemie, Germany) on the top of the culture medium in each well. The plates were incubated PXD101 concentration at 4°C for 1 h and then washed five times with tap water. The background optical NVP-HSP990 cost density was measured in the wells filled with culture medium, without the cells. The cellular material fixed with TCA was stained with 0.4% sulforhodamine B (SRB, Sigma, Germany) dissolved in 1% acetic acid (POCh, Gliwice, Poland) for 30 min. Unbound dye was removed by rinsing (4×) with 1% acetic acid. The protein-bound dye was extracted with 10 mM unbuffered tris base (POCh, Gliwice, Poland)

for determination of optical density (at 540 nm) in a computer-interfaced, 96-well microtiter plate reader Multiskan RC photometer (Labsystems, Helsinki, Finland). Each compound AZD9291 molecular weight in given concentration was tested in triplicates in each experiment, which was repeated 3–5 times. MTT assay This technique was applied for the cytotoxicity screening against mouse leukemia cells growing in suspension culture. An assay was performed after 72-h exposure to varying concentrations (from 0.1 to 100 μg/ml) of the tested agents. The compounds were dissolved in 10% DMSO to concentration of 1 mg/ml, and subsequently diluted in culture medium to reach

the required concentrations. DMSO, which was used as a solvent did not exert any inhibitory effect on cell proliferation. For the last 3–4 h of incubation 20 μl of MTT solution were added to each Ureohydrolase well (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; stock solution: 5 mg/ml). The mitochondria of viable cells reduce the pale yellow MTT to a navy blue formazan: the more viable cells are present in well, the more MTT will be reduced to formazan. When incubation time was completed, 80 μl of the lysing mixture was added to each well (lysing mixture: 225 ml dimethylformamide, 67.5 g sodium dodecyl sulfate, and 275 ml of distilled water). After 24 h, when formazan crystals had been dissolved, the optical densities of the samples were read on an Multiskan RC photometer at 570 nm wavelength. Each compound in given concentration was tested in triplicates in each experiment, which was repeated 3–5 times. The results of cytotoxic activity in vitro were expressed as an ID50—the dose of compound (in μg/ml) that inhibits proliferation rate of the tumor cells by 50% as compared to the control untreated cells. Acknowledgments This work is supported by Polish Ministry of Science and Higher Education, Grant No. N405 036 31/2655 and the Medical University of Silesia, Grant No. KNW-1-029/09.