Dihydrorhodamine 123 (solution) 

Dihydrorhodamine 123 (DHR 123) (solution) 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).
Solution Concentration: 10 mM

1. Solution preparation
1.1 Stock solution
Storage: Store at -20°C or -80°C in dark after aliquoting. Avoid repeated freezing and thawing.
1.2 Preparation of working solution
Dilute with cell culture medium (optimized according to the experiment). The corresponding stock solution can be diluted according to the actual situation. Note that if the solvent is DMSO, the cytotoxicity of DMSO must be considered, and a solvent control should be prepared; if the solvent is pure water, the working solution needs to be filtered and sterilized before adding cells.
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].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

346.39

C₂₁H₁₈N₂O₃

109244-58-8

O=C(OC)C1=CC=CC=C1C2C3=C(OC4=C2C=CC(N)=C4)C=C(N)C=C3

Room temperature in continental US; may vary elsewhere.

Please store the product under the recommended conditions in the Certificate of Analysis.

• [1]. Lydia M. Henderson et al. Dihydrorhodamine 123: a fluorescent probe for superoxide generation? Eur.J.Biochem. 217, 973-980.  [Content Brief]