The assay was performed according to the method of Skehan and co-workers . After incubation, the cells that were grown in 96-well plates (four wells per dose or concentration in
each of three independent experiments) were fixed with 10% trichloroacetic acid and stained for 30 min, when the excess dye was removed by washing with 1% acetic acid. The protein-bound dye was dissolved in 10 mM tris base solution for the determination of absorbance at 550 nm using Selleckchem SYN-117 a microplate reader (Victor, Wallac). Proliferation Assay The DNA synthesis and cell proliferation were measured using a 5-bromo-2-deoxyuridine (BrdU) assay (Roche Diagnostics GmbH, Mannheim, Germany). The cells were grown in 96-well plates (four wells per dose or concentration in each of three independent experiments) and BrdU labeling was performed according to the manufacturer’s instructions. The absorbance was measured at 550 nm using a microplate reader (Victor, Wallac). Clonogenic Assay After irradiation or drug treatment the cells were harvested by the trypsinization, seeded into 25-cm2 plastic tissue culture flasks (four flasks per dose or concentration in each of three independent experiments) at a suitable number for colony assay and incubated at 37°C for 7 days. This incubation period is appropriate since it represents more than six cell-doubling times. Moreover, the results of the colony
assay that was performed 14 days after irradiation did not show statistically significant differences in the cell inactivation level with respect to those obtained after Acalabrutinib 7 days . Therefore, in the combined treatments, during post irradiation incubation, the drugs were introduced after 4 days (without replating), and the cells were further incubated for 3 days. The cells were then fixed with methanol and stained with 10% Giemsa solution for the check details evaluation of the survival. Flow cytometry The cells
were grown in 25-cm2 plastic tissue culture flasks (four flasks per dose or concentration in each of two independent experiments). For the flow cytometric evaluation of the cell cycle status 1 × 106 cells were taken from each flask, washed with Phosphate Buffered Saline (PBS), fixed overnight with 70% cold ethanol and stained with PBS buffer that contained 50 μg/ml Propidium Iodide (PI) and Galactosylceramidase 50 μg/ml RNase. After the incubation for 30 min at room temperature, the cells were analyzed by the flow cytometry (Coulter EPICS XL; Beckman Coulter) using the XL SYSTEM II software. Statistical analysis Quadruplicate measurements were made during each experiment, while each experiment has been repeated three times, except for flow cytometry that was performed in two replicate experiments. All obtained data for viability, proliferation and survival assays were normalized to the untreated controls to obtain percentage of cells or surviving fraction.