Ros id total ros detection kit

Cell cycle analysis was performed as previously described (Sarkar Bhattacharya et al., 2019 (link)). Briefly, cells were dosed as indicated for 24 h. Cells were harvested by trypsin, washed in PBS with 1% BSA, and fixed in 70% cold ethanol. Pellets were treated with RNase A (Qiagen) and permeabilized with 0.1% TritonX-100 (Sigma). Cells were stained with propidium iodine (40 µg/ml final, Sigma) for 30 m at room temperature. Cells were analyzed using a FACSCalibur flow cytometer (Mayo Flow Cytometry Facility) with a minimum of 20,000 events and distribution percentage was calculated with CellQuest Pro software (BD Bioscience).
Total reactive oxygen species were measured by fluorescence detection using the ROS-ID Total ROS detection kit (Enzo Life Sciences) per manufacturer’s instructions. Briefly, 2,500 cells/well were seeded in a black 96-well plate and incubated overnight for attachment. Cells were dosed with quinacrine as indicated for 4 h. After dosing, cells were washed with PBS and ROS-ID dye was added for 30 m. Fluorescence was detected (488/520 nm excitation/emission) in a Synergy4 plate reader. Unstained and 0.5 mM hydrogen peroxide were used for negative and positive controls, respectively.

Cells grown on Matrigel-coated 35-mm glass bottom dishes were treated with indicated concentrations of BV for 1 h and then intracellular ROS generation was detected using ROS-ID Total ROS detection kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s instruction. In brief, oxidative stress detection reagent was added into BV-treated and -untreated iPSCs and the green fluorescent images were obtained using fluorescence microscope. In addition, fluorescence intensities were measured at Ex/Em = 490/525 nm using a SpectraMax3 microplate reader.

Quantification of Reactive Oxygen Species [ROS] namely hydrogen peroxide, peroxynitrite and hydroxyl radicals was achieved using the ROS-ID® Total ROS detection kit (ENZ-51011, ENZO Life Sciences, Inc.). All experiments were performed following the manufacturer's recommendations. ROS intracellular levels were examined using a 488-nm wavelength excitation on FACS VERSE. Acquired events were recorded using BD FACSuite software and analyzed using FlowJo v10.0.7, LLC software.

ROS activity was measured with the ROS-ID Total ROS detection kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s protocol. Briefly, cells were cultured for 4 days and collected. Subsequently, cells were washed and resuspended in ROS detection solution. Samples were incubated in the dark for 30 min and analyzed by flow cytometry at FL1 (490/525 nm) using a cytometer (Gallios, 3L10C, BECKMAN COULTER, Tokyo, Japan). The data were evaluated by triplicate measurements.

Total cellular ROS level in myoblast cells was measured with ROS-ID Total ROS detection kit (Enzo Life Science, Inc., NY, United States) 24 h after treatment. The cells were stained with Oxidative Stress Detection reagent and incubated at 37°C for 1 h. The stained cells were analyzed using a fluorescence microplate reader equipped with a 485/535 nm filter and observed under a fluorescence microscope.
Mitochondria were monitored by staining with MitoBright Green LT dye (Dojindo) and analyzed based on its fluorescence intensity. After staining the myoblast cells for 30 min, they were washed with PBS, followed by adding DMEM without phenol red. The cells were then observed over time under a fluorescence microscope. Images of fluorescence-stained cells at 24 and 72 h after treatment were captured under a GFP filter using a fluorescence microscope (Nikon Eclipse TC2000-S) equipped with a digital microscope camera (Nikon Ds-Ri2). The images were analyzed using the ImageJ software to measure mitochondrial density.

Intracellular ROS levels were measured using the ROS-ID® Total ROS Detection Kit (ENZO Life Sciences, Switzerland) according to manufacturer’s instructions with slight modifications. In brief, microglia were seeded in black clear bottom 96-well plates at a density of 5 × 10⁴ cells per well. Cells were allowed to adhere overnight and then incubated in serum-free medium, containing LPA in the absence or presence of the antagonists for indicated time periods. Thirty minutes before the end of each treatment, the cells were loaded with the ROS Detection Solution. Fluorescence intensity was measured with excitation and emission wavelengths of 485 nm and 535 nm, respectively.