Inducing Lagging Chromosomes and Micronuclei in PtK1 Cells

PtK1, H2B-PAGFP PtK1, and HEC1-GFP PtK1 [32 (link)] cells were cultured in Ham’s F-12 media (Invitrogen) supplemented with 10% Fetal Bovine Serum (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 14 mM sodium bicarbonate (Fisher Scientific), 1% antibiotic-antimycotic (Invitrogen), and maintained at 37° C in a humidified CO₂ incubator. For live cell imaging, cells were either grown on glass-bottom dishes and imaged using a stage top incubator (Tokai Hit) or were grown on sterilized coverslips inside 35 mm Petri dishes, transferred into a modified Rose chamber [33 (link)] with top coverslip, and imaged on a microscope stage heated by an air stream incubator (Nevtek). To induce LCs and MNi, cells were incubated in 20 μM STLC (S-Trityl-L-cysteine; Sigma-Aldrich) for 3 hours. The drug was then washed out by rinsing the cells 4 times with warm media. Cells were then re-incubated in fresh Ham’s F-12 media for 24 hours before immunostaining or live-cell imaging. For live-cell imaging, cells were placed in phenol-free L-15 media (Gibco) with 4.5 g/liter glucose. To induce DNA damage, 21 hours after STLC washout, cells were treated with 50 μg/ml Bleocin™ (antibiotic from Streptomyces verticillus; Calbiochem) for 3 hours and then fixed and immunostained as described in the next section.