Oxidative stress detection reagent

To directly monitor real time reactive oxygen species/reactive nitrogen species (ROS/RNS), a kit including an oxidative stress detection reagent (ENZO Life Sciences, Farmingdale, NY, USA) was used. Cells were exposed to the reagent for 35 min and then renin was dialyzed inside the cell and measurements of fluorescence intensity were made before and after intracellular renin (100 nM) administration using a wide filed fluorescence microscope equipped with standard green (490/525 nm) filter set.

Overnight cell cultures of the wild type and pulcherrimin mutant were inoculated in biological triplicate in 50 mL of LBGM supplemented with FeCl₃ at the final concentration of 0.2 mM. On the next day, 1 mL of each culture was harvested, washed with PBS three times, and incubated with the Oxidative Stress Detection Reagent from the ROS-ID® Total ROS Detection Kit (Enzo Life Sciences) according to the manufacturer’s protocol. Cells were imaged using fluorescence microscopy, and fluorescence quantification, data representation on a violin plot, and statistical significance were performed as described above. The Oxidative Stress Detection Reagent is a cell-permeable, non-fluorescent molecule that directly reacts with a variety of reactive oxygen species, including hydrogen peroxide, peroxynitrite, and hydroxyl radicals, producing green fluorescence result indicative of ROS presence. Therefore, there is a positive correlation between the intracellular levels of ROS and the fluorescence intensity observed.