Sytox® is a quick and easy way of quantifying death in your cell cultures. As cells become stressed and begin to die, the Sytox® signal goes up. The advantage of Sytox® is that it provides a very high signal. The disadvantage is that it does not give you any insight into the molecular causes of the cell death as an antibody would, for example.
Sytox® is an intercollating dye similar to SYBR® Green or DAPI. Like SYBR®Green, it is optimally read using a 485 nm/528 nm filter set on your plate reader. Sytox® is different than SYBR® Green in that it is only designed to stain cells that have their membrane compromised. Therefore, Sytox® should only stain dead or dying cells and not healthy cells. We have optimized our conditions for 96-well tissue culture plate format.
Through antibody staining (immunocytochemistry), we have confirmed that treatment with Sytox® does not significantly hurt the cells and is generally not much of a stress in and of itself during the hour or two of treatment. Although the listed protocol calls for only a 30 min incubation at 37ºC, we have noticed that the binding of Sytox® is increased as the temperature is lowered, thus increasing the signal without affecting the controls. We have also reduced the well-to-well variability by extending the incubation time. pH may also play a significant role in binding which deserves mentioning, but is something that we haven’t explored. We do not rinse the media from the wells prior to treatment with Sytox® because the dead or dying cells tend to detach from the bottom of the well. By rinsing before treatment, you would effectively wash away your signal. We always leave one well un-Sytoxed and stress-free so we can subtract the media background.
Sytox® is a quick assay that can save you time and money. You will want to confirm the Sytox® results with some other method, because there are situations of false signals. We confirm our results by fixing the cells and performing antibody staining. We have seen false negative results when cells have been treated with compounds that are also intercollating (like mitoxanthrone HCl, for example) which don’t allow the Sytox® to bind to the DNA. In wells that are extremely stressed, the DNases released by the dead cells in the well could degrade the DNA to the point that Sytox® has nothing to bind to. There may be other examples, but I wouldn’t worry too much about a false positive since not too many things in your cell culture are likely to fluoresce green enough to lead to such a problem.
Once your cells are fixed, the Sytox® signal can no longer be trusted. For later antibody analysis, we use Alexa 594 which is sufficiently far enough away from the Sytox® fluorescence to avoid bleed-over.