Visualizing Aberrant Metaphase Plates

To visualize aberrant metaphase plates, HDF_LT/hTERT cells at different passages (P16/P20/P24) were seeded at mid-confluence in eight-well BD Falcon™ CultureSlides (7 × 10³ cells for well) (BD Bioscience, Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Cells were arrested at pro-metaphase with 75 ng/mL of nocodazole (Sigma-Aldrich, Saint Louis, MI, USA) for 17 h plus 30 min of release, and prepared for immunofluorescence analysis as previously described in Pagotto et al. 2018 [6 (link)]. More than 150 images of metaphases were randomly acquired. Primary antibodies: anti alpha-tubulin (ab7291, Abcam, Cambridge Science Park, UK) and human anti-CREST (Antibodies Inc., Davis, CA, USA). Secondary antibodies: cross-adsorbed species-specific fluorescent secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA). DNA was detected using DRAQ5 (Cell Signaling Technology, Danvers, MA, USA). All the images were acquired using a Carl Zeiss LSM5 Pascal confocal laser scanning microscope (Carl Zeiss 63× Plan Neofluar oil-immersion objective, Oberkochen, Germany).