Chapter 21 Using Monoclonal Antibodies in Bromodeoxyuridine—DNA Analysis

The utility of bromodeoxyuridine (BrdUrd) as a marker for cell cycle traverse studies has been substantially increased by the introduction of monoclonal antibodies (mAb) against BrdUrd incorporated into cellular DNA. These antibodies are useful as immunological reagents to stain cells containing BrdUrd fluorescently so that the intensity of fluorescence is proportional to the amount of incorporated BrdUrd. The intensity of fluorescence is great enough to permit easy microscopic or flow cytometric (FCM) analysis of BrdUrd incorporation. The cytokinetic utility of the BrdUrd labeling has been further increased by the technique of simultaneous measurement of DNA content and the amount of incorporated BrdUrd. The BrdUrd–DNA assay is based on a procedure for simultaneously staining cells with dyes that fluoresce at different wavelengths. The procedure requires that the DNA is partially denatured to expose incorporated BrdUrd to a specific antibody. Denaturation is necessary because antibodies developed so far bind only to BrdUrd in single-stranded DNA. Green fluorescence from the fluorescein-conjugated antibody is a measure of BrdUrd incorporation.