Hydroethidine [Dihydroethidium] *CAS 104821-25-2*

Hydroethidine [Dihydroethidium] stock solution:
Make 5-10 mM DMSO stock solution. Note: The unused DMSO stock solution should be aliquoted into single use vial and stored at -20 °C. Keep awayfrom light.

Hydroethidine [Dihydroethidium] dye working solution:
Make the dye working concentration of 5 – 20 µM in a physiological buffer (such as PBS, HBSS, HEPES). Note: The optimal working concentration for your application must be empirically determined.

1. Add equal volume (such as 100 µL of the cells in growth medium) of the dye working solution to the cells, and incubate the cells at RT or 37°C for 5 to 60 minutes.
   
   
2. Determine the baseline fluorescence intensity of a sample of the loaded cells prior to exposing the cells to experimental inducements.
   
   
3. Negative controls should be assessed as follows:
   
   
   1. Examine the fluorescence of cell-free mixtures of dye and buffer/media with and without the inducer. In the absence of extracellular esterases and other oxidative enzymes, the gradual increase in fluorescence over time may be related to spontaneous hydrolysis, atmospheric oxidation, and/or light-induced oxidation.
      
      
   2. Examine the fluorescence of untreated (control) loaded cells that have been maintained in growth medium or buffer. In healthy cells, oxygen radicals are eliminated by cellular enzymes and/or natural antioxidants. Following the dye-loading recovery period, healthy cells should exhibit a low level of fluorescence that is relatively stable for the duration of the experiment; however, a gradual increase (due to auto-oxidation) or decrease (due to loss of dye from cells or photobleaching) in fluorescence may be observed. In the absence of any stimulus or inducement, a burst of fluorescence in healthy, untreated cells could indicate progress to cell death or some other oxidative event.
      
      
4. Positive controls may be stimulated with tert-butyl hydroperoxide (TBHP) to a final concentration of ~100 µM (increase or decrease dose based on the sensitivity and response of the cells).