DNA labeling in living cells

Background: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. Methods: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. Results: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP Conclusions: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission. (c) 2005 Wiley-Liss, Inc.