Cellular ROS detection assay (Abcam PLC, Cambridge, MA, USA) was used to measure hydroxyl, peroxyl, and other ROS activity within the cell following the manufacturer's instructions. Cells were seeded into each well and cultured for at least 4 h before measurements. Cells were washed once with PBS supplemented with 2′, 7′-dichlorofluorescein diacetate (DCFDA) or Deep Red Fluorescence. After incubation at 37°C for 1h, cells were resuspended in PBS in the presence of freshly prepared 10% FBS followed by another 30 minute culture at 37°C. The cells were analyzed on FACS Calibur flow cytometer (BD Biosciences). Relative fluorescence intensities were analyzed and histograms generated using Cell Quest.
Cellular ros detection assay
Manufactured by Abcam
Sourced in United States
The Cellular ROS Detection Assay is a fluorescence-based kit designed to measure the levels of reactive oxygen species (ROS) in living cells. The assay utilizes a cell-permeable fluorogenic probe that becomes fluorescent upon oxidation by various ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals. The intensity of the fluorescent signal is proportional to the amount of ROS present in the sample, providing a quantitative measure of cellular oxidative stress.
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4 protocols using cellular ros detection assay
1
Cellular Reactive Oxygen Species Assay
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Intracellular ROS Detection in HCEC-1CT Cells
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To detect intracellular ROS, the 2′,7′-dichlorofluorescein diacetate (DCFDA), Cellular ROS Detection Assay was used, according to manufacturer’s protocol (Abcam). The non-fluorescent, cell-permeable DCFDA diffuses into cells, where it is deacetylated by celluar esterases to 2′,7′-dichlorodihydrofluorescin. In the presence of ROS, the dichlorodihydrofluorescin is oxidized to the highly fluorescent 2’,7’-DCF and further detected by fluorescence spectroscopy. 5 × 103 HCEC-1CT cells/96-well were grown for 24 h to 70–80% confluence. Cells were treated with 25 ng/ml TNF-α, IL-6 or IL-8 for 15 min, 30 min, 1 h, 2 h or 3 h. Subsequently, cells were washed twice with PBS, 100 μl of 25 μM DCFDA in HBSS was added and incubated for 30 min. Cells were washed with cold PBS, 100 μl HBSS was added and cells were incubated for 10 min at 37°C. Subsequently, fluorescent cells were measured on a Chameleon V microplate reader (Hidex, Ex/Em = 485/535 nm). Measurements were carried out in triplicates.
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Intracellular ROS Detection in HCEC-1CT Cells
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Cellular ROS Quantification Assay
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