Quantitative lamin B1 disassembly assay. (A) High-throughput automated time-lapse wide-field microscopy of HeLa cells stably coexpressing H2B-mCherry and GFP–lamin B1. A single cell entering mitosis is shown. The last interphase frame marks time point 0. For each condition, several hundred mitotic events were acquired with a time-lapse of 2 min, and up to eight different experimental conditions were imaged in parallel. (B) H2B-mCherry signals were used for automatic tracking of nuclei and for classification-based annotation of mitotic stages. In addition, error-corrected mitotic classification results were used to determine mitotic progression (sum of pro-, prometa-, and metaphase duration; Held et al., 2010). (C) GFP–lamin B1 intensities were measured at the chromatin and in the cytoplasm throughout mitosis. Segmentation of the H2B-mCherry signal was used to define the chromatin region (in) and by dilation the cytoplasmic region (out). (D) Automated analysis of lamin B1 disassembly. The difference between the GFP–lamin B1 intensity at chromatin (in) and in the cytoplasm (out) was determined during mitosis and normalized to the difference during interphase. Using a mathematical model, disassembly durations were determined as the time in which the intensity difference changed from 95 to 5% (green bars). a.u., arbitrary units. Bars, 10 µm.