Dihydroethidium

Protocol tips

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This product should be kept under argon. It is both light and air sensitive. It will oxidize to ethidium on storage unless kept under argon.

Upstream tips
This product should be kept under argon. It is both light and air sensitive. It will oxidize to ethidium on storage unless kept under argon.

Publication protocol

Fluorometric measurement of superoxide anion production
Superoxide anion production was detected using the dihydroethidium (DHE) probe. 5 × 105 cells/well were seeded on 6-well tissue culture plates and allowed to adhere for 24 h in a 5% CO2 incubator at 37 0C. Thereafter, cells were treated with Oxa and Mel in their respective medium. Further, cells were stained or incubated with DHE solution (2 μg/ml) for 20 min. DHE dye incorporated into the cells and subsequently oxidized to the fluorescent ethidium cation by superoxide anion, allowing the cation to bind nuclear DNA with an extensive enhancement of fluorescence at 530 nm when excited at 485 nm [23]. The fluorescence signal was detected as proportional to superoxide production. The DHE fluorescence integrated density was analyzed with Image J software (1.50 version, NIH, USA). The 3–4 images were captured using NIS elements software provided by the Nikon Ts Eclipse inverted microscope at 20x magnification. Maximum projections were generated from focus planes and images were displayed with equal pixel intensity. Results were expressed as percentage of DHE stained positive cells.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Dihydroethidium below.

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