The CellSimple™ Cellular Reactive Oxygen Species (ROS) Detection Assay Kit is a fluorescent assay designed for use with the CellSimple™ Cell Analyzer. It detects overall ROS levels in living cells. The kit contains the non-fluorescent fluorescein derivative DCFH-DA, a common ROS inducer tert-butyl hydroperoxide (TBHP), Loading Buffer, and 20X PBS wash buffer. DCFH-DA diffuses into cells during pre-incubation and is deacetylated by cellular esterases to form DCFH, which can be rapidly oxidized to the highly fluorescent DCF by cellular ROS. The median fluorescent intensity (MFI) of DCF fluorescence in the green channel (525/45 nm) is used to quantify overall levels of cellular ROS.
The CellSimple™ Cellular Reactive Oxygen Species (ROS) Detection Assay Kit is expected to detect reactive oxygen species in living cells across all species. A cell number titration is recommended to be performed when developing an assay with your cells of interest.
The CellSimple™ Cell Analyzer is a benchtop instrument that utilizes a disposable thin-film cassette and a combination of a 488 nm laser, two photomultiplier tubes (525/45 nm and 561 nm LP filters), Coulter Principle-based cell measurements, and on-board software to provide easy-to-run applications and data analysis. Data acquisition occurs within approximately 10 seconds per test. The instrument relies on disposable cassettes for sample handling, which alleviates the need for flow cell cleaning and fluidics maintenance and the instrument is small enough to be portable between the lab bench and the hood. Applications include quantitative assessments of cell viability, apoptosis, other labeled antibody markers and single and multiplexed bead-based assays for protein and cellular analysis.
Reactive oxygen species (ROS) include a variety of highly reactive oxidant molecules and free radicals that are derived from molecular oxygen. ROS are generated as a result of both normal cellular metabolism and environmental factors including air pollutants or cigarette smoking. The major ROS of physiological significance are superoxide anions (•O2-), hydroxyl radicals (•OH), and hydrogen peroxide (H2O2) (1-3). Low to moderate ROS levels play important roles in normal physiological processes including vascular tone regulation, oxygen sensing, immunological reaction, regulation of translation, and signal transduction. High ROS concentrations can lead to adverse modifications of cellular components, such as carbohydrates, lipids, proteins, and DNA. Many of these modifications have been linked to diseases including atherosclerosis, diabetes, cancer, and neurological disorders. As a result, regulation of reducing and oxidizing (redox) state is not only critical for cell health, but is also an attractive therapeutic target for many diseases (1-3).
2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) is a non-fluorescent fluorescein derivative used as a non-ionic, non-polar, cell membrane permeable dye. After crossing the cell membrane, DCFH-DA is hydrolyzed by intracellular esterases to a non-fluorescent dichlorodihydrofluorescein (DCFH). DCFH is relatively more polar and cell membrane-impermeable and thus accumulates in cells. In the presence of cellular ROS, DCFH is oxidized to the highly fluorescent dichlorofluorescein (DCF). The fluorescent intensity of DCF is proportional to the overall cellular ROS level (4,5). These fluorescent signals can be detected using the Open Flow Cytometry application on the CellSimple™ Cell Analyzer.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
CellSimple is a trademark of Cell Signaling Technology, Inc.