CHO AA8 cells (24 (link)) were grown in monolayer or suspension culture in αMEM supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin and 100 U/ml penicillin. Cells were counted and analyzed on a Coulter® Multisizer II. The plating efficiency of AA8 and other repair-proficient cell lines was ∼90%, and that of 51D1 was ∼70%; the doubling times for AA8 and 51D1 were ∼13 and ∼16 h, respectively.
To determine the cell cycle distribution of each cell line 5 × 105 cells were treated with 10 µg/ml BrdUrd for 20 min at 37°, fixed with 70% ethanol and stained with fluorescein isothiocyanate (FITC)-conjugated anti-BrdUrd antibody (BD Biosciences) and propidium iodine to determine the proportion of cells in each phase and DNA content, respectively. Fluorescence measurements of each sample were made on a FACscan (Becton Dickinson) and the data analyzed using Cell Quest software.
To determine the cell cycle distribution of each cell line 5 × 105 cells were treated with 10 µg/ml BrdUrd for 20 min at 37°, fixed with 70% ethanol and stained with fluorescein isothiocyanate (FITC)-conjugated anti-BrdUrd antibody (BD Biosciences) and propidium iodine to determine the proportion of cells in each phase and DNA content, respectively. Fluorescence measurements of each sample were made on a FACscan (Becton Dickinson) and the data analyzed using Cell Quest software.
