Reactive oxygen species (ROS) play a crucial role in physiological and pathological processes such as cell growth, proliferation, development, differentiation, aging, and apoptosis. In order to accurately detect the level of intracellular ROS, various ROS reactive oxygen species assay kits have been developed. This article introduces the principle, usage method, and precautions of ROS reactive oxygen species assay kit to help researchers conduct better experimental research.
1、 Principle of ROS Reactive Oxygen Species Kit
The ROS reactive oxygen species kit mainly uses fluorescent probes to detect intracellular ROS levels. These fluorescent probes, such as DCFH-DA or CellROX Green, have weak or no fluorescence themselves, but can freely penetrate the living cell membrane and enter the cell. In cells, these probes are significantly enhanced in fluorescence intensity after being oxidized by ROS. Therefore, fluorescence signals can be detected by instruments such as fluorescence microscopy, laser confocal microscopy, fluorescence spectrophotometer, or flow cytometer to determine the level of intracellular ROS.
2、 Usage of ROS Reactive Oxygen Species Kit
Cell preparation: According to experimental requirements, lay or collect cells to ensure their healthy state and sufficient quantity.
Drug induction (optional): Remove the cell culture medium, add an appropriate amount of drug diluted with suitable buffer or serum-free medium, and incubate in a 37 ℃ cell culture box in the dark to induce ROS production. The specific induction time depends on the drug characteristics and cell type.
Probe loading: According to the instructions, dilute the fluorescent probe with serum-free culture medium and add it to the cells. Incubate it in a 37 ℃ cell culture box in the dark for a period of time to allow the probe to fully enter the cells and be oxidized by ROS.
Cell cleaning: Wash cells with serum-free culture medium to remove probes that have not entered the cells.
Fluorescence detection: Using instruments such as fluorescence microscope, laser confocal microscope, fluorescence spectrophotometer, or flow cytometer, fluorescence signals are detected at specific excitation and emission wavelengths to determine the level of intracellular ROS.
3、 Precautions
Storage conditions: The ROS reactive oxygen species kit should be stored in a dark environment at -20 ℃ to avoid probe failure caused by light and high temperature.
Operating standards: Strictly follow the instructions to avoid contamination and incorrect operation that may result in inaccurate experimental results.
Instrument calibration: Before using fluorescence detection instruments, instrument calibration should be performed to ensure the accuracy and reliability of the detection results.
Experimental control: It is recommended to set up positive and negative controls to verify the reliability and accuracy of the experimental results.
In summary, the ROS reactive oxygen species assay kit is a rapid, simple, and sensitive method for detecting intracellular ROS levels. By strictly following the instructions and precautions, researchers can accurately measure the level of intracellular ROS, providing strong support for further research on the role of ROS in cellular physiological and pathological processes.
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