Cellular reactive oxygen species detection assay kit red fluorescence

Cellular ROS generation was measured with the Cellular Reactive Oxygen Species Detection Assay Kit (Red Fluorescence), which was purchased from Abcam (Cambridge, UK) [23 (link)]. Cells were seeded at 1×10⁵ cells/100 μL per well in a 96-well plate. ROS Red Working Solution (100 μL) was added and incubated in a 37°C/5% CO₂ incubator for 60 min. Cells were then treated with test compounds for 60 min to induce ROS production. An increase in the fluorescence intensity at an excitation wavelength of 535 nm and an emission wavelength of 590 nm was monitored in bottom read mode.

Reactive Oxygen Species (ROS) levels were measured for PEO1^S, PEO4^R, PEA1^S, PEA2^R, PEO14^S, PEO23^R, and FT4 cells using the Cellular Reactive Oxygen Species Detection Assay Kit (Red fluorescence, ab 186027, Abcam, USA). Briefly, 10,000 cells/well of each cell line were plated into Thermo Scientific Nunc MicroWell 96-well optical-bottom plates with polymer base (Cat#165305, Thermo Scientific, USA). Cells were allowed to attach for 48 hours. The manufacturer’s protocol was followed for the ROS assay, and time-resolved fluorescence was monitored at Ex/Em = 520/605 nm with bottom read mode on a SpectraMax M5 Multi-mode microplate reader (Molecular Devices, USA). ROS levels were measured for PEO4 WT and PEO4 KO using a flow cytometry-based assay. Single-cell suspensions were treated with 20 μM DCFDA and the fluorescence (Ex/Em = 485/535 nm) was measured. Flow cytometry was performed at the University of Illinois at Chicago RRC facility using CyAn flow cytometer (Beckman Coulter Inc., Fullerton, CA). All data were analyzed by Summit software (Beckman Coulter Inc., Fullerton, CA). N-Acetyl-L-cysteine (NAC) was purchased from Sigma (MO, USA), (#1009005).