Dihydroethidium (Hydroethidine)

It has been done. The problem is that plate readers are less sensitive than microscopes, with far less signal-to-background difference. It is worth trying, but first optimize concentrations and loading times with control cells, use a plate with little to no autofluorescence, and possibly optimize the gain setting in order to get the best signal possible. But don't expect the same sensitivity, even with optimization.

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This is not recommended as the two dyes overlap in the emission wavelength. There are other ROS reagents available in different wavelengths, such as CellROX Deep Red, which emits in the far-red range (665 nm), or dihydroethidium, which is emits in the visible red range (620 nm).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

• Dissolve Dihydroethidium (DHE) in high quality, anhydrous DMSO or DMF up to 10 mM and store in aliquots at -20 to -80°C under argon or nitrogen. DHE oxidizes more readily in solution and is very sensitive to light. Avoid repeated freeze/thaws.
• Treat adherent cells to induce reactive oxygen species (ROS).
• Wash 2-3 times with a physiological buffer (PBS, HBSS).
• Incubate about 30 minutes at 37°C in about 1-20 µM DHE diluted in buffer.
• Wash 2-3 times more with buffer.
• Image or analyse using filters appropriate for excitation/emission = 302, 518/605 nm.