DNA double labelling with IdUrd and CldUrd for spatial and temporal analysis of cell proliferation and DNA replication

A procedure was developed that very effectively distinguishes between IdUrd and CldUrd incorporated in the DNA of cell nuclei and chromosomes. For double staining we used the, rat anti-BrdUrd monoclonal antibody from Sera-lab that binds specifically to CldUrd and BrdUrd but not to IdUrd, in combination with the mouse anti-BrdUrd monoclonal antibody from Becton Dickinson. This antibody binds to all three halogenated deoxyuridines, but when the nuclei are washed in TRIS buffer with a high salt concentration the antibodies linked to CldUrd-labelled DNA are removed. When analysing the effect of the deoxyuridines on the cell cycle we found that the growth kinetics of Chinese hamster cells were not changed by adding IdUrd or CldUrd for 30 min at a concentration of 10 μm, whereas adequate double labelling required only 2 min pulses. The effectiveness of the technique was demonstrated in two model experiments. The first test concerned the assessment of cell recruitment in the central areas of slow-growing clones, after addition of fresh medium. The second experiment focussed on the spatial resolution of the method. Double-labelled metaphase chromosomes showed interspersed green and red replication bands with a spacing corresponding with medium resolution Giemsa banding patterns.