312 mg/ml) of Alamar Blue (Resazurin, Sigma Aldrich Co. St. Louis, MO, USA) was added to each www.selleckchem.com/products/VX-765.html well. The absorbance was measured using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter®), and the drug effect was quantified as the percentage of control absorbance at 570 and 595 nm. The absorbance of Alamar Blue in culture medium is measured at a higher wavelength and lower wavelength. The absorbance of the medium is also measured at the higher and lower wavelengths. The absorbance of the medium alone is subtracted from the absorbance of medium plus Alamar

Blue at the higher wavelength. This value is called AOHW. The absorbance of the medium alone is subtracted from the absorban‘ce of medium plus Alamar Blue at the lower wavelength. This value is called AOLW. A correction factor R0 can be calculated from AOHW and AOLW, where R0 = AOLW/AOHW. The percent Alamar Blue reduced is then expressed as follows: % reduced = ALW − (AHW × R0) × 100. Cultured human lymphocytes were plated at a concentration of 0.3 × 106 cells/ml and incubated for 24 h with different concentrations of PHT (0.25, 0.5, 1.0, 2.0, and 4.0 μM) and then mixed with low-melting point agarose. Doxorubicin (0.5 μM) was used as a positive

control. The alkaline version selleck screening library of the comet assay (single cell gel electrophoresis) was performed as described by Singh et al. (1988) with minor modifications (Hartmann and Speit, 1997). Slides were prepared in duplicate, and 100 cells were screened per sample (50 cells from each duplicate slide), using a fluorescence microscope (Zeiss) equipped with a 515–560 nm excitation filter, a 590 nm barrier filter, and a 40× objective. Cells were scored visually according to tail length into five

classes: (1) class 0: undamaged, without a tail; (2) class 1: with a tail shorter than the diameter of the head (nucleus); (3) class 2: with a tail length 1–2× the diameter of the head; (4) class 3: with a tail longer than 2× the diameter of the head; (5) class 4: comets with no heads. Two different but complementary parameters were employed: Damage index (DI) and damage frequency (DF). DI is based on migration length and on the amount Thalidomide of DNA in the tail, and it is considered a sensitive DNA measure. A value (DI) was assigned to each comet according to its class, using the formula: DI = (0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4), where n = number of cells in each class analyzed. The damage index ranged from 0 (completely undamaged: 100 cells × 0) to 400 (with maximum damage: 100 cells × 4). On the other hand, DF represents the percentage of cells (tailed cells) with DNA damage ( Speit and Hartmann, 1999). Naturally synchronized human peripheral blood lymphocytes were used with more than 95% of cells in the G0 phase (Bender et al., 1988 and Wojcik et al., 1996). Short-term lymphocyte cultures, at a concentration of 0.3 × 106 cells/ml, were initiated according to a standard protocol (Preston et al., 1987).