Dihydroethidium

Dihydroethidium (DHE, HE, Hydroethidine, PD-MY 003) is a cell-permeable blue fluorogenic probe used for detecting intracellular superoxide radical anion.

1.1 Preparation of the stock solution
Dissolve 1 mg of Dihydroethidium in 0.31 mL of DMSO to obtain 10 mM of Dihydroethidium.
Note: It is recommended to store the stock solution at -20°C or -80°C away from light and avoid repetitive freeze-thaw cycles.
1.2 Preparation of Dihydroethidium working solution
Dilute the stock solution in serum-free cell culture medium or PBS to obtain 1-10 μM of Dihydroethidium working solution.
Note: Please adjust the concentration of Dihydroethidium working solution according to the actual situation.
Cell staining
2.1 For suspension cells: Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
For adherent cells: Discard the cell culture medium, and add trypsin to dissociate cells to make a single-cell suspension. Centrifuge at 1000 g at 4°C for 3-5 minutes and then discard the supernatant. Wash twice with PBS, 5 minutes each time.
2.2 Add 1 mL of Dihydroethidium working solution, and then incubate at room temperature for 30 minutes.
2.3 Centrifuge at 400 g at 4°C for 3-4 minutes and then discard the supernatant.
2.4 Wash twice with PBS, 5 minutes each time.
2.5 Resuspend cells with serum-free cell culture medium or PBS, and then detect by fluorescence microscope or flow cytometer.