Cell experiment:

Dihydrofluorescein diacetate is used at a final concentration of 5 μM for the cell-free studies. The excitation wavelength is 488 nm and the emission wavelength is 521 nm, the same as the excitation and emission wavelengths of the confocal microscope. All fluorescence measurements are performed on room temperature solutions. Human umbilical vein endothelium at first passage are cultured to confluence on 48 well plates. Cells are ished with PBS, then PBS with glucose, pH 7.4, is added to the cells. Dihydrofluorescein diacetate is added at a final concentration of 20 mM. Wells without cells are also loaded with fluorescent probes to serve as controls. Fluorescence measurements are made on a fluorescent platereader every 10 min at Exλ 488 nm and Emλ 510 (bandpass 5 nm) and 538 nm (bandpass 10 nm). The Emλ 510 nm and Emλ 538 nm values are added together[1].

Dihydrofluorescein diacetate is a fluorimetric probe mainly used for oxidative stress measurements, in both cell-free systems and cellular models.

Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies. It is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. Dihydrofluorescein diacetate demonstrates fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium[1]. Dihydrofluorescein diacetate is able to detect the presence of ROS in mitochondria. Dihydrofluorescein diacetate fluorescence was sharp and delineated thin filaments which corresponded in all details to TMRM-stained mitochondria. It enters mitochondria and reacts with ROS released in the matrix[2]. Dihydrofluorescein diacetate could be an useful and quantitative method for measuring the oxidative potential of nanoparticle-treated cells[3].

[1]. Hempel SL, et al. Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123. Free Radic Biol Med. 1999 Jul;27(1-2):146-59. [2]. Diaz G, et al. Mitochondrial localization of reactive oxygen species by dihydrofluorescein probes. Histochem Cell Biol. 2003 Oct;120(4):319-25. [3]. Aranda A, et al. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay: a quantitative method for oxidative stress assessment of nanoparticle-treated cells. Toxicol In Vitro. 2013 Mar;27(2):954-63.