A novel deep red/low infrared fluorescent flow cytometric probe, DRAQ5NO, for the discrimination of intact nucleated cells in apoptotic cell populations

Background: The linking of intracellular metabolism of anticancer drugs wit h cellular response is problematic. We describe a new probe for cellular in tegrity, based upon a structure which has the additional potential to act a s a substrate for cytochrome P450-dependent bioreductive metabolism. DRAZ5N O is an N-oxide modified anthraquinone with optimal fluorescence excitation maxima compatible with He-Ne (633 nm) and Kr-Ar (647 nm) lasers. Methods: DRAQ5NO-loading and Annexin V binding was monitored using dual-las er how cytometry (488 nm/633 nm wavelengths) in human lymphoma cultures und ergoing anticancer drug- (etoposide; VP-16) induced apoptosis. Results: DRAQ5NO gave an Em(lambda max) of 700.5 nm but retains DNA binding potential with an emission wave-length red-shift of similar to 12 nm. The agent showed reduced cytotoxicity and a limited capacity to accumulate with in cells compared with the non-N-oxide form that shows a high nuclear targe ting capacity in intact cells. DRAQ5NO/Annexin V provides for a positive di scrimination between intact cells, membrane-compromised cells, cellular deb ris, and early stage apoptotic cells. Conclusions: The spectral properties of DRAQ5NO allow for the use of visibl e range fluorochromes and differential excitation in multilaser systems for tracking apoptotic populations with implications for the measurement of bi oreductive potential in complex tumour populations simultaneously undergoin g physiologically or drug-induced-apoptosis. (C) 2000 Wiley-Liss, Inc.