Quantifying Somatic Nuclei Replication Patterns

Somatic nuclei replication patterns were assessed as described [58 (link)]. Briefly, CHO nuclei replicating in egg extract were pulse-labelled with 25 μM Cy3-dUTP (GE Healthcare) for 2.5 min. Nuclei were isolated and fixed in 4% paraformaldehyde and spun down onto poly-l-lysine coverslips. Total DNA was stained with Hoechst 33258 and coverslips were mounted with Vectashield mounting medium and sealed. Images were acquired using a cooled camera (CoolSNAP HQ; Photometrics) on a restoration microscope (DeltaVision Spectris; Applied Precision) built around a stand (Eclipse TE200; Nikon) with a 100× 1.4 NA Plan Apo lens (Nikon). Images were taken every 0.25 μm, and 22 optical sections were recorded for every nucleus. Three-dimensional datasets were deconvoluted using the constrained iterative algorithm software (SoftWoRx; Applied Precision), and images were analysed in the Open Microscopy Environment (www.openmicroscopy.org). Timing patterns for 20 nuclei at each data point were classified as described [58 (link)] in three independent experiments.