Dcf da

Intracellular generation of ROS was determined using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA; Molecular Probes, Thermo Fisher Scientific Inc.). The dye that integrated into the cells was deacetylated by intracellular esterases. Upon oxidation, DCF-DA is converted to highly fluorescent 2,7-dichlorofluorescein (17 (link)). Briefly, cells were cultured at 37°C overnight in 6-well plates and then treated with GSP in the presence or absence of N-acetyl cysteine (NAC) for 4 h. The cells were stained with 5 µM DCF-DA in serum-free medium for 15 min and removed from the plate with trypsin-EDTA (Gibco, Thermo Fisher Scientific, Inc.). The fluorescence intensity of the cells was determined by flow cytometry with an excitation wavelength of 480 nm and an emission wavelength of 525 nm (BD Biosciences). Data were analyzed using CellQuest Pro software (version 4.0, BD Biosciences).

Intracellular ROS were assayed through the oxidation of 2′, 7′‐dichlorofluorescein diacetate (DCFDA, Molecular Probes).24 Briefly, mRPTCs were incubated with fresh DCFDA (10 μmol/L) in serum‐free medium for 30 minutes at 37°C. DCFDA fluorescence was measured, in 96‐well plates, using a microplate reader, at an excitation wavelength of 485 nm and emission wavelength of 530 nm. ROS production was expressed in arbitrary units, corrected for protein concentration (arbitrary units/per mg protein).
ROSstar 550 (LI‐COR Biosciences) is a cell‐permeable hydrocyanine probe which is initially non‐fluorescent but becomes fluorescent after oxidation by ROS.25 The ROSstar 550 assay is specific for oxygen radicals, in particular for superoxide and hydroxyl radicals. The mRPTCs were incubated with 50 μmol/L ROSstar 550 for 30 minutes at 37°C, washed 2 times with PBS, and the fluorescence was immediately read, in the plate reader at excitation of 540 nm and emission of 560 nm. The data were normalized by the protein concentration in each well.