Intracellular glutathione gsh detection assay kit

The intracellular ROS and GSH levels were assessed by flow cytometry, using DCF-DA and Intracellular glutathione (GSH) Detection Assay Kit (ab112132; Abcam, Cambridge, UK), respectively. 200,000 cells/well were seeded in two separate 6-well plates, one for ROS and one for GSH detection. After a 24 h treatment with increasing concentrations of the brine extract, the medium was removed and 1 mL of 10 μΜ DCF-DA (20 mM stock in DMSO) in PBS was added in each well of the ROS plate, and incubated in the dark at 37 °C for 45 min. After this incubation period, cells were trypsinized, centrifuged (1200 rpm, 5 min, 4 °C) and resuspended in 250 μLPBS. At the same time, when the 24 h treatment period was ended, cells of the GSH plate were trypsinized immediately, centrifuged (1200 rpm, 5 min, 4 °C) and GSH detection was performed according to the manufacturer’s instructions. Briefly, cells were resuspended in 1 mL PBS with 5 μL of Green dye and incubated in the dark at 37 °C for 30 min. After this, the cells were washed with 1 mL PBS to remove the excess dye, centrifuged (1200 rpm, 5 min, 4 °C) and resuspended in PBS. The cells were then submitted to flow cytometric analysis using a FACScan flow cytometer (Becton Dickinson, NJ, USA) with excitation and emission length at 490/520 nm for ROS and for GSH detection.

Intracellular GSH levels were measured using Intracellular glutathione (GSH) Detection Assay Kit (Abcam) and the fluorescence intensity was detected by flow cytometry at E_x/E_m = 490/520 nm.