Sequential Labeling of Replicating DNA

Asynchronously growing DT40 cells were sequentially labeled for 20 min with 25 μM IdU and for 20 min with 25 μM CIdU. Dideoxycytidine (ddC) treated cells were exposed to 2 mM ddC just before the CldU treatment. At the end of the labeling period (40 min), cells were placed in ice cold 1 × PBS (1 volume of cells for 2 volumes of 1 × PBS) and centrifuged at 250 g for 5 min at 4°C, washed in ice-cold PBS, and re-suspended in PBS to a final concentration of 1 × 10⁶ cells/ml. 3 μl of the cell suspension was spotted onto clean glass Superfrost slides and lysed with 7 μl of 0.5% SDS in 200 mM Tris–HCl (pH 5.5) and 50 mM EDTA (5 min, at room temperature). Slides were tilted at 15° to horizontal, allowing the DNA to run slowly down the slide. Slides were then air dried and fixed in 3:1 methanol/acetic acid, and stored at 4°C before immunolabelling. IdU, CldU revelations and analysis were performed as described (20–22 (link)), with minor modifications: the DNA was denatured for 30 min in 2.5 N HCl, and CldU was detected using rat anti BrdU (ABD Serotec, Raleigh, NC) at 1/750. A stretching factor of 2.6 for conversion from μm to kb was applied, as previously described for the method in (23 (link)). Slides were mounted in 10% 1× PBS and 90% glycerol, kept at −20°C and imaged using a Nikon C1-si confocal microscope.