INTERACTIONS OF INTERCALATING FLUOROCHROMES WITH DNA ANALYZED BY CONVENTIONAL AND FLUORESCENCE LIFETIME FLOW-CYTOMETRY UTILIZING DEUTERIUM-OXIDE

Deuterium oxide (D2O) has been shown in previous studies to increase b oth the fluorescence Lifetime and fluorescence intensity of propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid struc tures, We have used spectroscopic analysis and conventional and phase- sensitive now cytometry to compare changes in PI and EB fluorescence i ntensity and lifetime bound to DNA and fixed Chinese hamster ovary (CH O) cells in the presence of D2O vs. phosphate-buffered saline (PBS), S pectroscopic and now cytometric studies showed a twofold enhancement o f fluorescence intensity of PI and EB bound to fixed CHO cells in D2O and a 5 ns increase in PI and EB fluorescence lifetimes in D2O. The fl uorescence lifetime of HL-60 cells stained with PI of EB was found to be 1-2 ns different from that of CHO cells, indicating that the lifeti me of these fluorochromes is sensitive to chromatin configuration in d ifferent cells types. Apoptotic subpopulations of HL-60 cells had a si gnificantly reduced fluorescence lifetime compared to nonapoptotic sub populations. Results indicate that different chromatin states, or diff erences in the structures of PI and EB, lead to alterations in the flu orescence intensity and fluorescence Lifetime of these intercalating p robes. (C) 1996 Wiley-Liss, Inc.