Ninety-six-well culture dishes were inoculated with PG100 cells at a density of 1 × 106 cells Tenofovir price per ml. Following incubation for 24 h, the cells were then

incubated in DMEM containing 100 or 250 μg/ml of unmodified or biotransformed green tea extract or EGCG. After 24 h of incubation, the comet assay was performed on the exposed cells. The cell positive control was the cells non-treated with the tea samples. To detect DNA damage, the alkaline comet assay was performed on the cell suspensions using a modified version of the method described by Singh, Mccoy, Tice, and Schneider (1998). Briefly, 20 μl of the cell suspension was mixed with molten 0.5% low-melting-point agarose (Promega Co., Madison, WI, USA) and spread on agarose-precoated microscope slides. The slides were immersed overnight in freshly prepared cold lysing solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris, 2% sodium salt N-lauryl sarcosine, pH 10, with 1% Triton X-100 and 10% dimethyl sulphoxide; all from Sigma–Aldrich) at 4 °C. After incubation, the slides were washed in cold PBS (Invitrogen Life Technologies) for 30 min. Subsequently, the cells were exposed to alkaline buffer (1 mM EDTA and 300 mM NaOH, pH 13.4) at 4 °C, for 40 min to allow DNA unwinding and expression of alkali-labile sites. Everolimus clinical trial Electrophoresis was then conducted in the same solution at 4 °C for 20 min

at 25 V and 300 mA. After electrophoresis, the slides were neutralised (0.4 M Tris, pH 7.5), stained with 40 μl EtBr (20 mg/ml) and analysed with a fluorescence Etomidate microscope (Eclipse E400; Nikon, Melville, NY, USA), using the Komet 5.5 image analysis system (Kinetic Imaging, Nottingham, UK). One hundred randomly selected cells (50 from each of two replicate slides) were evaluated from each sample, and the mean olive Tail moment was determined. Tail moment (TM) is defined as the product of the fraction of the total DNA in the tail and the mean distance of migration in the tail and is calculated by multiplying tail intensity/sum comet intensity by the tail’s centre of gravity peak position. A higher percentage of tail DNA signifies a higher level of DNA damage. Ninety-six-well

culture dishes were inoculated with PG100 cells at a density of 10 × 108 cells per well. Four replicate wells were inoculated for each sample tested. After incubation at 37 °C, in an atmosphere of 5% CO2 and 100% relative humidity for 24 h, cells were incubated in media containing pre-defined concentrations (from 50 to 250 μg/ml) of unmodified or biotransformed green tea extract or EGCG. Positive controls (untreated cells) were also performed. After incubation for 48 h, the cultures were assayed for cancer-related gene expression. The cells were collected, and total RNA was isolated using an RNeasy® tissue kit (QIAGEN). Single-stranded cDNA was synthesised using a High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s protocol.