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Get ready for a night full of darkness as Nightfall finally comes to Minecraft..How to detect and validate inhibitory activity of SYTOX Green and SYTOX Red.
During the eukaryotic cell death process the occurrence of SYTOX Green (SG) or SYTOX Red (SR) positive cells and nuclei in cell cultures is useful as an indicator of apoptotic cell death. However, the presence of such chromatin positive cells in cell cultures could be due to a variety of other causes, for instance permeabilization of the cell membranes for both dyes occurs in necrotic cell death. Thus, it is important to have a rapid method for validating the inhibitory activity of the experimental compounds. The method described in this chapter is based on the observation that the stable form of SG, which is the SG pentamethine cyclopentadithiopene, is not released by live cells. The method was applied to screen antineoplastic compounds. We showed that the cytotoxic effect of vincristine, a well-known tubulin inhibitor, was due to SR, and not to SG. Then we measured the drug sensitivity to vincristine of neuroblastoma cells in the presence of actinomycin D. We showed that the previously recorded IC50 of vincristine in neuroblastoma cells with actinomycin D was increased by 20-fold in the presence of the cytotoxic action of vincristine was dependent on SR, but not SG. This validates the use of the SR cell death assay to detect and validate the antineoplastic action of compounds.