Heating and co2 incubation system

Live-cell imaging was adapted from^{45 (link)}. HeLa H2B–mCherry and tubulin–eGFP cells were transfected with siRNA oligonucleotides in 8-well µ-slide chambers (Ibidi). The cells were imaged for 48 h starting at 24 h post-transfection (approx.), using a Plan-Apochromat 20 × NA 0.8 objective and a 488-nm and 561-nm diode lasers on a LSM 5 live confocal microscope (Zeiss) equipped with a heating and CO₂ incubation system (Ibidi). ZEN software (Zeiss) was used to acquire images from seven 3.6-µm-spaced optical z-sections at various positions every 3 min. Then, single position files were generated from the maximum intensity projections in ZEN and converted into image sequences with free licensed AxioVision software (LE64; V4.9.1.0). Segmentation, annotation, classification and tracking of cells progressing through mitosis were performed using the Cecog analyser (http://www.cellcognition.org/software/cecoganalyzer)^{32 (link)}. The subsequent analysis was performed in Microsoft excel and GraphPad Prism. Three independent experiments were performed.

HeLa cells expressing H2B-mCherry and EGFP-α-tubulin were transfected with siRNA oligonucleotides in eight-well μ-slide chambers (Ibidi) and, after 24 h, were imaged for 48 h in a LSM 5 live confocal microscope (Zeiss) equipped with a heating and CO₂ incubation system (Ibidi). Seven 3.6-μm-spaced optical z-sections at various positions every 3 min were acquired with a Plan-Apochromat 20× NA 0.8 objective and a 488-nm and 561-nm diode lasers controlled by ZEN software. For the analysis, maximum intensity projections in Z were generated in ZEN for every position and converted into temporal image sequences with the free licensed AxioVision software (LE64; V4.9.1.0). Afterward, segmentation, annotation, classification, tracking of cells during mitosis, and extraction of galleries with the identified cell tracks were performed using the Cecog Analyzer (http://www.cellcognition.org/software/cecoganalyzer) (Held et al, 2010 (link)) The percentage of tracks with persistently misaligned metaphase chromosomes was identified as clearly isolated chromosomes separated from the metaphase plate in two or more consecutive frames, and visually determined. Microsoft Excel and GraphPad Prism were used for data analysis from more than 100 cell tracks per condition in three independent experiments.