Dihydrorhodamine 123

Dihydrorhodamine 123 (DHR 123) is a non-fluorescent reactive oxygen species (ROS) indicator. Dihydrorhodamine 123 is oxidized to fluorescent Rhodamine 123 (HY-D0816) within cells in the presence of reactive oxygen species and it localizes in mitochondria (Ex/Em = 515/536 nm). In Vitro:1. Solution preparation
1.1 Preparation of stock solution
Solvent: DMSO
Concentration: 10 mM.
Storage: Store at -20°C or -80°C in dark after aliquoting. Avoid repeated freezing and thawing.
1.2 Preparation of working solution
Dilute to 1-10 µM with cell culture medium (optimized according to the experiment).
Note: The working solution should be prepared and used immediately. Keep it away from light.

2. Cell staining
2.1 Treat cells with inducers or inhibitors of reactive oxygen species. Note: If treating cells with oxidizing agents, rinse cells three times with buffer or culture medium to remove the compounds before staining to avoid direct oxidation of the dye by the compound.
2.2 Dilute Dihydrorhodamine 123 dye stock solution in serum-free culture medium at a final concentration of 5 μM.
2.3 Remove medium from the cells and replace with medium containing dye.
2.4 Incubate for 30 minutes at 37°C.
2.5 Detect by fluorescence microscopy or flow cytometry. Washing is not required, but can be performed to reduce background for microscopy.

In the presence of 10 μM Dihydrorhodamine 123 (DHR 123) the stimulation of the neutrophil NADPH oxidase by the addition of 50 nM phorbol 12-myristate 13-acetat (PMA) resultes in an increase in the rate of rhodamine generation. The fluorescent intensity of the cells, in the presence of 10 μM Dihydrorhodamine 123, increases with time following the addition of 50 nM PMA. In the presence of 10 μM Dihydrorhodamine 123, induced HL60 cells show a sustained increase in fluorescence following the addition of 50 nM PMA^[1].