細胞內總 ROS 活性測定

細胞內總 ROS 活性測定為評估氧化應激抑製劑和活化劑試劑的效力提供了一個很好的篩選選項，並將有助於確定氧化應激如何調節不同的細胞內途徑。可評估細胞內 ROS 活性的總體水平，並可使用流式細胞儀進行分析。

Background Reactive oxygen species (ROS) are natural byproducts of the normal metabolism of oxygen and play important roles in cell signaling. However, during oxidative stress-related states, ROS levels can increase dramatically. The accumulation of ROS results in significant damage to cell structures. The role of oxidative stress in cardiovascular disease, diabetes, osteoporosis, stroke, inflammatory diseases, a number of neurodegenerative diseases and cancer, has been well established. ICT’s Intracellular Total ROS Activity Assay provides a good screening option for assessing the potency of oxidative stress inhibitor and activator reagents, and will help to determine how oxidative stress modulates varied intracellular pathways. This kit assesses the overall level of intracellular ROS activity, but does not identify the specific reactive oxygen molecule(s) generated by the oxidative stress event. The key reagent in the assay is a proprietary dye called Total ROS Green. This dye quickly penetrates membrane structures and accumulates within the cell. In the presence of ROS, the non-fluorescent Total ROS Green dye molecule is oxidized by all various iterations of ROS molecular forms. In the oxidized state, the Total ROS Green dye molecule acquires fluorescence properties that enable its detection by flow cytometry as an indicator of the relative level of intracellular ROS activity.

Target ROS

Excitation / Emission 490 nm / 520 nm

Method of Analysis Flow cytometry

Sample Type Cell culture

Storage Total ROS Green and DMSO at ≤-20°C, Assay Buffer at ≤2-8°C

Country of Origin United States

Protocols

1. Prepare samples and controls in 1X Assay Buffer or buffer of choice (0.5 mL at a cell density of 5 x 105 – 1 x 106 cells/mL).

2. Reconstitute Total ROS Green with 100 µL DMSO to produce a 500X stock concentrate.

3. Dilute 1:10 by adding 900 µL 1X Assay Buffer to the 500X stock Total ROS Green to produce a 50X working solution.

4. Add Total ROS Green to each sample at 1:50. For example, add 10 µL into 490 µL cultured cells.

5. Incubate 1 hour at 37°C.

6. Treat cells with test compounds for desired period of time. Keep cells protected from light.

7. Analyze with a flow cytometer. Total ROS Green excites at 490 nm and emits at 520 nm.

Kit Contents

Total ROS Green, lyophilized, 1 vial, #6688 DMSO, 200 µL, 1 vial, #6689 Assay Buffer, 10X, 15 mL, #686 Kit Manual

Citations (1)

Kaproń B, Czarnomysy R, Wysokiński M, Andrys R, Musilek K, Angeli A, Supuran CT, Plech T. 1,2,4-Triazole-based anticonvulsant agents with additional ROS scavenging activity are effective in a model of pharmacoresistant epilepsy. J Enzyme Inhib Med Chem. 2020 Dec;35(1):993-1002. doi: 10.1080/14756366.2020.1748026. Full Text