Applications and caveats on the utilization of DNA-specific probes in cell-based assays

Abstract

To perform cell-based assays using fluorescence as the readout there is a fundamental need to identify individual cellular objects. In the majority of cases this requires the addition of a DNA dye or so-called nuclear counterstain and these have become integral to assay design. End-point assays can use live or fixed cells and thus it is beneficial if such reagents are cell membrane-permeant. Further, membrane-permeant DNA dyes can open new opportunities in dynamic real time assays with caveats according to the impact of their interaction with the chromatin in live cells. As cell-based assays offer information on the in vitro toxicity of treatments, cell viability has become a basic readout and cell membrane-impermeant fluorescent DNA-specific dyes can provide this information. In the case of both nuclear counterstaining and viability reporting, it is beneficial if the DNA dyes employed are suitably spectrally separated to permit multi-color experimental design. Methods will be described for these two important assay readouts.