2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) is one of the most widely used methods for measuring intracellular ROS generation This dye is a stable compound that readily diffuses into cells and hydrolyzed by intracellular esterase to DCFH which is trapped within cells. In the presence of H2O2 or low-molecular weight hydroperoxides, peroxidases can oxidize DCFH2 to 2′-7′-dichlorofluorescein (DCF). The level of peroxide produced can be estimated by an increase in DCF fluorescence at 530 nm when the sample is excited at 485 nm17, 18. In the present study, cells were treated with 100 ?M of the 4 -HBTC for 24- 72 hours. The cells were incubated with 50 ?l DCFH-DA (10 ?M) for 30 min. Then, after twice washing with PBS amount of DCFH-DA fluorescence was detected using flow cytometry.
Antioxidant enzymes assay
SOD activity in the cell lysate was assayed using a method based on the capability of the enzyme to inhibit the autoxidation of pyrogallol. Briefly, 1 ml Tris-EDTA buffer 5 mM (pH=8.0) was mixed with 50 ?l of the cell lysate supernatant. The unit was blanked at 420 nm and then 50 ?l of pyrogallol (0.2 mM) was added to the above solution and swiftly the absorbance of samples was evaluated at 420 nm every 15 s, up to 10 minutes. The inhibition of pyrogallol autoxidation is commensurate with the activity SOD present in the sample. Enzyme inhibitory capacity is determined as one unit of SOD19, 20. The enzyme activity was reported as IU/mg of protein.
Catalase activity was determined by Aebi method, that monitoring the decomposition of hydrogen peroxide21. 1 ml Tris-EDTA buffer 5 mM (pH=7.0) and 25 ?l hydrogen peroxide (10 mM) added to cuvette. Absorbance decrease based on the decomposition of H2O2 was measured at a wavelength of 240 nm for 30 seconds.
Determination of total thiol content
Total -SH groups are indicators of free radical damages. This sensitive factor decreases in oxidative damage conditions. Thiol groups were evaluated using DTNB (5, 5?-dithiobis- 2- nitrobenzoic acid) as the Ellman’s reagent.This reagent reacts with the SH groups and produces a yellow color. Its absorbance was measured with spectrophotometrically at 412 nm. 22. To perform this assay, 1 ml Tris EDTA buffer (pH 8.6) was added to 50 ?l cell lysate and the absorbance of the sample was read at 412 nm against Tris-EDTA buffer alone (A1). Then, 25 ?l DTNB reagents (10 mM in methanol) was added to the cuvette mixture and after 15 min (stored at laboratory temperature), the sample absorbance was read again (A2). The absorbance of DTNB reagent was also read as a blank (B). Total thiol concentration (mM) was measured via the following equation:
Total thiol concentration (mM)= (A2-A1-B) × 1.07/0.05 × 13.6
Measurement of MDA level
The concentration of MDA as a marker of lipid peroxidation was measured via thiobarbituric acid reactive substances (TBARS) assay in the KG1a cells lysate. This technique is based on the reaction of two molecules of thiobarbituric acid (TBA) with one molecule of MDA. The ultimate soluble including the substances which are responsible for the pink color23. Absorption of the samples was read at 532 nm using a UV–vis spectrophotometer.