Fluorescence enhancement of DNA-bound TO-PRO-3 by incorporation of bromodeoxyuridine to monitor cell cycle kinetics

Background: The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycl e. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual- laser flow cytometry has been investigated. Methods: Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is re gistered in combination with the fluorescence emission of the intercalating dye propidium iodide (PI) as a total DNA stain to give bivariate DNA/BrdUr d histograms. By the low concentration of only 0.3 mu M TO-PRO-3, BrdUrd de tection is optimized, and undisturbed total DNA content by PI can be detect ed as well. TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser. Results: In order to understand the binding of TO-PRO-3, energy transfer fr om PI to TO-PRO-3 has been measured as well as the influence of an external DNA binding dye such as Hoechst 33258 with Adenine-Thymine (AT) binding sp ecificity. Cell cycle studies of human SCL-2 keratino-cytes and mouse 3T3 c ells prove the method to be as generally applicable as the classical BrdUrd /Hoechst quenching technique, but without need for ex-pensive ultraviolet l aser excitation. No BrdUrd sensitivity could be found for the similar dyes TO-PRO-1 and YO-PRO-3, whereas TO-PRO-5 and YOYO-3 showed only very little sensitivity to BrdUrd labeling as compared with TO-PRO-S. Conclusions: Cell cycle studies of mammalian cells can be done by dual-lase r flow cytometry without the need for ultraviolet lasers by using the BrdUr d-dependent fluorescence enhancement of TO-PRO-S. Total DNA content can be measured simultaneously using PI. Cytometry 37:221-229, 1999. (C) 1999 Wile y-Liss, Inc.