Eclipse 80i microscope

Cells were cultured on chamber slides and then treated with alisertib (62.5 nmol/L) for 24 h before incubating with extraction buffer (20 mmol/L HEPES, pH 7.5, 20 mmol/L NaCl, 5 mmol/L MgCl₂, 0.5% Nonidet P-40), on ice for 20 min, then fixed in 4% NBF for 15 min, and permeabilized in 0.5% Triton-X100/PBS for 10 min at room temperature. Cells were blocked in 5% normal goat serum in TBS for 1 h at room temperature and then incubated with anti- pH2AX^S139 diluted in block overnight at 4°C and then with goat-anti-mouse Dylight 488-conjugated secondary antibody for 1 h at room temperature. Immunofluorescent images were acquired using a Nikon Eclipse 80i microscope (Melville, NY) and digital camera with Metamorph software v. 7.7 (Molecular Devices, Sunnyvale, California) using identical exposure times. The number of nuclei with ≥ 10 pH2AX^S139-positive foci was enumerated in five different fields for each treatment condition and the mean percentage of nuclei with ≥ 10 pH2AX^S139 foci ± SE was calculated. Statistical analysis was performed with GraphPad Prism using Wilcoxon matched-pairs signed rank test; P < 0.05 was considered significant.