2 ,7 -Dichlorodihydrofluorescein

FIGURE 1.13 Absorbance measurement of 2, 7 -dichlorodihydrofluorescein (DCDHF), a peroxynitrite-sensitive dye, as a function of nitrite (1 mM) and NADH (1 mM) introduction to a solution containing 100 pM DCDHF and 30mU/mL NR under ambient (a) and nitrogen saturated conditions (b). As can be seen, die absorbance of DCDHF increases upon nitrite and NADH introduction only under an oxygen atmosphere, indicative of peroxynitrite production. (Reprinted with permission from Elsevier Publishing [117].)... 

A method based on inhibition of ABAP-induced oxidation of a fluorogenic probe, 2, 7-dichlorodihydrofluorescein diacetate (H2-DCF-DA), commonly used for detection of production of reactive oxygen species, has been proposed (V2). Oxidation of the probe can be monitored either spectrophotometrically or fluori-metrically. A drawback of the method is the necessity of hydrolysis of the ester because only the deesterified from can be oxidized. The rate of hydrolysis depends on the hydrolytic activity of the samples measured. A modification consisting of chemical hydrolysis of H2-DCF-DA before measurement is impractical because 2,7 -dichlorodihydrofluorescein oxidizes rapidly. 

As an example, the assay commonly used to measure the direct generation of ROS by nanoparticles is based on the conversion by ROS of the 2,7-dichlorodihydrofluorescein dye into a fluorescent product, 2,7-dichlorofluorescein. There is also a range of fluorescent probes that measure NM-induced ROS production inside the cells, in different intracellular compartments (e.g., dihydrorhoda-mine-1,2,3 in the mitochondria, 2,7-dichlorodihyydrofluorescein diacetate in the cytoplasm, dihydroethidium bromide in the nucleus) [59]. They are all relatively easy to use for quantification in a fluorimeter, multiwall plate reader, or by flow cytometry, but a potential drawback of all these assays is the background caused by particles as well as the fluorescence quenching effects that need to be controlled and taken into account to reliably measure the free radical production [59, 60]. 

Fig. 5. Reactive oxygen species (ROS) detection in rat Schwann cells in vivo. Cells were cultured in Dulbecco s modified Eagle medium supplemented with 10% fetal bovine serum and either 5 vaM (A) or 30 vaM glucose (B). They were then incubated for 45 min in phosphate buffer saline, pH 7.5, containing 10 M of the ROS-sensitive molecular probe 5- (and 6)-chloromethyl-2, 7 -dichlorodihydrofluorescein diacetate (CM-H DCFD A) and viewed by fluorescence microscopy.

Materials. Arabidopsis seedlings were cultured according to the procedures described previously.9 The components of artificial acid rain are H2SO4, HNO3 and HC1 in a ratio of 1 1 1.1 Catalase (CAT, bovine liver) and 2 , 7 -dichlorodihydrofluorescein diacetate (H2DCFDA) were from Sigma (St. Louis, MO, USA). 

Afri, M., Erimer, A.A., Cohen, Y., 2004. Active oxygen chemistry within the hposomal bilayer Part IV locating 2, 7 -dichlorofluorescein (DCF), 2, 7 -dichlorodihydrofluores-cein (DCFH) and 2, 7 -dichlorodihydrofluorescein diacetate (DCFH-DA) in the lipid bilayer. Chem. Phys. Lipids 131, 123—133. 

Chen, X., Zhong, Z., Xu, Z., Chen, L., Wang, Y., 2010. 2, 7 -Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement forty years of application and controversy. Eree Radic. Res. 44, 587—604. 

Wrona, M., Patel, K., Wardman, P., 2005. Reactivity of 2, 7 -dichlorodihydrofluorescein and dihydrorhodamine 123 and their oxidized forms toward carbonate, nitrogen dioxide, and hydroxyl radicals. Free Radic. Biol. Med. 38, 262—270. 

Figure 7.2 (a) Cigarette smoke extract (CSE)-induced reactive oxygen species were quenched by resveratrol (Res). Human alveolar epithelial (A549) cells were treated with 1.0-5.0% CSE with or without lOgM resveratrol for 24 h, and ROS were measured by flow cytometry using 5- (and 6)-carboxy-2, 7 -dichlorodihydrofluorescein diacetate... 

Qin, Y. Lu, M. Gong, X. Dihydrorhodamine 123 is superior to 2,7-dichlorodihydrofluorescein diacetate and dihydro-rhodamine 6G in detecting intracellular hydrogen peroxide in tumor cells. Gell Biol. Int. 2008, 32, 224-228. 

J. R. Manning, P. Birch-Machin, M. A. Implications of using the fluorescent probes, dihydrorhodamine 123 and 2, 7 -dichlorodihydrofluorescein diacetate, for the detection of UVA-induced reactive oxygen species. Free Radical Res. 2011,45,115-122. 

Two fluorescent probes dihydroethidium (DHE) and dichlorodihydrofluorescein (DCFH) are used for superoxide detection in biological systems [54]. Both assays are subjected to some drawbacks such as the oxidation of cytochrome c, the dismutation of superoxide in the presence of DHE [79], and the interaction of DCFH with hydrogen peroxide, hydroperoxides, or peroxynitrite. Particularly big enhancement of DCF formation due to the oxidation of cytochrome c is observed when cytochrome is released from mitochondria during cell death... 

Keller, A., Mohamed, A., Drose, S., Brandt, U., Fleming, I., Brandes, R.P., 2004. Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species. Free Radic. Res. 38, 1257—1267. 

Extracellular reduced glutathione could completely attenuate the cristobalite (particle diameter 1.2 on)-induced expression of monocyte chemo-tactic protein-1 and macrophage inflammatory protein-2 mRNAs by a murine alveolar type II line, whereas TNF-a mRNA levels were unaltered (Barrett et al. 1999). Using the oxidant sensitive dye, 6-carboxyl-27 -dichlorodihydrofluorescein diacetate di(acetoxymethyl ester) treatment of this cell line with cristobalite (18 ftg/cnf = 100 pg/ial) and TNF-a (1 ng/ml) resulted in the production of reactive oxygen species, which could be inhibited with extracellular GSH treatment. In the case of cristo-balite-induced reactive oxygen species, inhibition was also achieved with an anti-TNF-a antibody. 

ROS scavenging ability of ERG was tested using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) solution-based chemical assay and a 2,2,7,2-dichlorodihydrofluorescein diacetate (DCFH-DA) HL-60 cell line-based assay. The cell-based fluorescent DCFH assay enables detection of antioxidant molecules which can penetrate cell membranes and inhibit ROS production in living cells. The antioxidant activity was determined by TPA-stimulated control cells with and without FRG. FRG showed strong antioxidant activity in both systems [64]. 

Trayner, I. D. Rayner, A. P. Freeman, G. E. Farzaneh, F. Quantitative multiwell myeloid differentiation assay using dichlorodihydrofluorescein diacetate (H2DCF-DA) or dihydrorhodamine 123 (H2R123). J. Immunol Methods 1995,186, 275-284. 

Crow, J. P. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitrite in vitro imphcations for intracellular measrrrement of reactive nitrogen and oxygen species. Nitric Oxide 1997,1, 145-157. 

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