Dichlorofluorescin diacetate is hydrolysed by cellular esterases to dichlorofluorescin (DCFH) and is then oxidized to a fluorescent product dichlorofluorescein (DCF) primarily bu H2O2. Dihydrorhodamine (DHR 123) is by far the most-used probe for measurement of intracellular H2O2. DHR 123 is oxidized directly to rhodamine 123, which is excitable at 488 and emits at 515 nm in the same emission range as DCF and FITC (Rothe et al., 1988). Publications describe its use in human neutrophils, human eosinophils, HL60 cells, rat mast cells, guinea pig neutrophils, cultured chondrocytes, rat brain, rat renal proximal tubular cells, mesangial cells and L929 cells. Combinations of DHR123 with surface receptor analysis cell viability using propidium iodide, and calcium indicators demonstrate how the probe can be used for simultaneous measurements.
DHR 123 enters the cells as a freely permeable dye which is converted to rhodamine 123 and subsequently localized in the mitochondria. The conversion from the non-fluorescent to the fluorescent molecule is entirely dependent upon oxidation products and does not require enzymatic catalysis. Once oxidized, the probe is identical to rhodamine 123, a common laser dye. One significant advantage of the DHR probe is that the oxidation product, rhodamine 123, remains essentially within the cell, unlike the oxidation product DCF, which has a strong tendency to leak from cells and requires careful controls to monitor leakage.