Andrew Niles1, Min Zhou2, Laurent Bernad2, Mark McDougall2 and Dan Lazar1
1Promega Corporation, Madison, WI 53711 and 2Promega Biosciences, San Luis Obispo, CA 93401.
Cell-based models continue to be critical tools for cancer research. Therefore, the assessment of cytotoxic or cytostatic effects remains an obligate experimental activity. Although numerous enzyme activity chemistries exist for measuring cytotoxicity, biomarker degradation may cause underestimation of actual cytotoxicity and limit the utility of these activity measures when ascertained with long-term drug exposures (72 hours or more).
We have developed a cell-impermeant probe, CellTox-Green™ dye, which labels the DNA from cells with compromised membranes. The quantum efficiency (i.e., brightness) of this dye increases dramatically upon DNA binding, producing a fluorescent signal which can be measured using standard “green” fluorometry wavelengths (excitation 485nm/emission 530nm). Additionally, the dye is non-toxic and stable, allowing for introduction of the probe directly into the culture medium or compound dilutions prior to cell dosing. This “no step” addition feature allows for the measurement of cytotoxicity in real time or at a convenient endpoint. Furthermore, the dye is fully compatible with luminescent measures, allowing for same-well, multi-parametric assessment of the cytotoxic phenotype.
Lastly, proliferative or anti-proliferative effects (relative to control) can be revealed by the introduction of a lytic detergent which enables the normally impermeant dye to bind to all DNA in the assay well. Herein, we detail our efforts to characterize and multiplex this dye when applied to suspension and adherent cell lines treated with agents which cause either primary or secondary necrosis in a 72hr time course.