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.
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.
Jurkat cells were stained with ICT’s Intracellular Total ROS Activity Assay (kit #9144) for 1 hour, and then treated with a negative control (left histogram) or 100 μM tert-Butyl hydroperoxide, a reactive oxygen-inducing agent used to create the positive control (middle histogram), for 30 minutes at 37°C. Cells were read on the FL1 channel of an Accuri C6 flow cytometer. The median fluorescence intensity (MFI) of stained cells in the negative control was 11,875 in FL1-A (left: Negative), whereas the treated population had a MFI value of 419,067 (middle: Positive), which is an increase of more than 35-fold. The effect of tert-Butyl hydroperoxide on intracellular total ROS activity is easily visible when the samples are overlaid in a single plot (right-most histogram, black: Negative; right, red: Positive). Data courtesy of Dr. Kristi Strandberg, ICT, 224:76.
• Target: ROS
• Method of Analysis: Flow Cytometer
• Types of Samples: cell culture
• Excitation/Emission: 490 nm / 520 nm
• Storage: Total ROS Green and DMSO at ≤-20°C, Assay Buffer at ≤2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping
• Total ROS Green, lyophilized, 1 vial, #6688
• DMSO, 200 µL, 1 vial, #6689
• Assay Buffer, 10X, 15 mL, #686
• Kit Manual