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

Abstract

The linking of intracellular metabolism of anticancer drugs with cellular response is problematic. We describe a new probe for cellular integrity, based upon a structure which has the additional potential to act as a substrate for cytochrome P450-dependent bioreductive metabolism. DRAQ5NO is an N-oxide modified anthraquinone with optimal fluorescence excitation maxima compatible with He-Ne (633 nm) and Kr-Ar (647 nm) lasers. DRAQ5NO-loading and Annexin V binding was monitored using dual-laser flow cytometry (488 nm/633 nm wavelengths) in human lymphoma cultures undergoing anticancer drug- (etoposide; VP-16) induced apoptosis. DRAQ5NO gave an Em(lambdamax) of 700.5 nm but retains DNA binding potential with an emission wavelength red-shift of approximately 12 nm. The agent showed reduced cytotoxicity and a limited capacity to accumulate within cells compared with the non-N-oxide form that shows a high nuclear targeting capacity in intact cells. DRAQ5NO/Annexin V provides for a positive discrimination between intact cells, membrane-compromised cells, cellular debris, and early stage apoptotic cells. The spectral properties of DRAQ5NO allow for the use of visible range fluorochromes and differential excitation in multilaser systems for tracking apoptotic populations with implications for the measurement of bioreductive potential in complex tumour populations simultaneously undergoing physiologically or drug-induced apoptosis.