Axioplan 2 microscope

Mitotic cells were washed with warm media by centrifuging at 300 × g for 3 min. Cells were suspended in 500 µl of warmed swelling buffer (40% complete media + 60% deionized water). Samples were incubated in a 37°C water bath for 15–18 min. Swollen cells were fixed by adding 1 ml 3:1 methanol: acetic acid and then incubated for 10 min. The cells were pelleted for 5 min at 250 × g and then washed with 1 ml fixative and pelleted once more. The cell pellets were resuspended in 100–200 µl fixative, and then 40–50 µl of cell suspension was dropped from a height of 60 cm onto a 22-mm² coverslip that was cleaned with 95% ethanol and wiped with acetic acid. The coverslips were immediately placed inside a 150-mm plastic culture dish on top of wet filter paper. The lid was left off, and the coverslips were allowed to dry in the humidified chamber. Once dried, coverslips were stained with 4′,6-diamidino-2-phenylindole (DAPI) (100 ng/ml) and SYBERGold nucleic acid dye (1:20,000). Slides were imaged with a Zeiss Axioplan II microscope using a 100× objective, Hamamatsu Orca II camera, and Metamorph software. At least 200 mitotic spreads were scored for each sample. If an individual cell spread had more than 10 single chromatids, then the cell was scored as fatigued.