Proliferation index, mitotic duration and chromosome missegregation were scored by visual inspection of time-lapse movies. Proliferation was calculated as the ratio of all live cells in the last movie frame (24 h) divided by all live cells from the first movie frame. Mitotic duration was defined as the interval from prometaphase onset until anaphase onset for all cells entering mitosis within the first 12 h of the experiment, and determined based on the chromatin morphology. The incidence of chromosome missegregation was calculated by dividing the number of anaphase cells with lagging or bridged anaphase chromosomes by the total number of anaphase cells.
DNA labelling specificity was quantified 2 h after adding the different dyes by automated image analysis using the open-source CellCognition software20 (link). Cell nuclei were automatically segmented in five consecutive image frames per experimental condition by local adaptive thresholding in the H2B-mCherry channel. Mitotic cells and dead cells were excluded from analysis based on automated classification by supervised machine learning. The mean fluorescence was then measured in the far-red channel. To calculate the nucleo/cytoplasmic fluorescence ratio, a cytoplasmic region was defined for each cell as a 5 pixel wide rim around the nuclear segmentation mask, spaced at a distance of 1 pixel. Extracellular background fluorescence was manually measured and subtracted from intracellular mean fluorescence measurements. The nucleo/cytoplasmic fluorescence ratio was first calculated for individual cells to derive the mean nucleo/cytoplasmic fluorescence ratio of the cell population. The mean and s.e.m. was then calculated for each dye condition based on three independent biological replicates. Fluorescence cross-talk from the H2B-mCherry channel was very low (<2% of the signal detected in 500 nM SiR–Hoechst-treated cells), as assessed by measuring fluorescence intensity in dimethylsulfoxide-treated control cells.
DNA labelling specificity was quantified 2 h after adding the different dyes by automated image analysis using the open-source CellCognition software20 (link). Cell nuclei were automatically segmented in five consecutive image frames per experimental condition by local adaptive thresholding in the H2B-mCherry channel. Mitotic cells and dead cells were excluded from analysis based on automated classification by supervised machine learning. The mean fluorescence was then measured in the far-red channel. To calculate the nucleo/cytoplasmic fluorescence ratio, a cytoplasmic region was defined for each cell as a 5 pixel wide rim around the nuclear segmentation mask, spaced at a distance of 1 pixel. Extracellular background fluorescence was manually measured and subtracted from intracellular mean fluorescence measurements. The nucleo/cytoplasmic fluorescence ratio was first calculated for individual cells to derive the mean nucleo/cytoplasmic fluorescence ratio of the cell population. The mean and s.e.m. was then calculated for each dye condition based on three independent biological replicates. Fluorescence cross-talk from the H2B-mCherry channel was very low (<2% of the signal detected in 500 nM SiR–Hoechst-treated cells), as assessed by measuring fluorescence intensity in dimethylsulfoxide-treated control cells.
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