Dual Labeling of DNA Synthesis for Replication Monitoring

Cells were labeled with 100 µM iododeoxyuridine (IdU) for 10 min and then labeled with100 µM chlorodeoxyuridine (CldU) for 20 min for mock experiment. For monitoring DNA synthesis during replication stress, cells were labeled with IdU (100 µM) for 10 min, followed by exposure to CldU (100 µM) coupled with hydroxyurea (5 mM for HCT115 and 0.5 mM for human fibroblasts) for 1 h. DNA fibers were spread as described previously (29 (link)) and stained with primary antibodies (mouse anti-BrdU/IdU from BD Bioscience; rat anti-BrdU/CldU from Accurate Chemical) and fluorescence-conjugated secondary antibodies (Alexa Fluor 488-anti-rat and Texas Red-conjugated anti-mouse from Invitrogen). Fibers were imaged using Zeiss Axio Imager M2 and measured using AxioVision software (x64 version 4.8.3.0).