ROS, also known as reactive oxygen species, is a chemical substance with oxidative activity produced within cells, including hydrogen peroxide, superoxide anions, and so on. ROS can also be produced under normal physiological conditions, but when it is produced excessively or cleared insufficiently, it can cause oxidative stress to cells, leading to cell damage and disease.
Detection principle
The most widely used method for detecting intracellular ROS is the DCFH-DA fluorescent probe method. DCFH-DA (dichlorofluorescein diacetate) is an indicator probe that does not have fluorescence itself, but can freely penetrate the cell membrane. Once it enters the cell, it will be hydrolyzed into DCFH by intracellular esterases. Due to DCFH's inability to penetrate the cell membrane, fluorescent probes accumulate within the cell. The intracellular ROS can oxidize non fluorescent DCFH to generate fluorescent DCF, and the fluorescence intensity is proportional to the ROS level. Detect fluorescence at a maximum excitation wavelength of 480nm and a maximum emission wavelength of 525nm using equipment such as fluorescence microscopy, flow cytometry, or laser confocal microscopy
Testing steps
1、 Loading probes
Cells with short stimulation time (usually within 2 hours): First load the probe, and then stimulate the cells with reactive oxygen species positive control or drugs of interest.
Cells with longer stimulation time (usually 6 hours or more): First, stimulate the cells with a positive control of reactive oxygen species or a drug of interest, and then load the probe.
1. In situ loading probe (applicable only to adherent cells)
2. Collect cells and load probes (applicable to adherent cells and suspended cells)
2、 Testing
In situ loading probe method: direct observation with laser confocal microscope, or detection with fluorescence spectrophotometer, fluorescence enzyme-linked immunosorbent assay or flow cytometer after collecting cells.
Collect cells and load probes: detect with a fluorescence spectrophotometer, fluorescence enzyme-linked immunosorbent assay reader, or flow cytometer, or directly observe with a laser confocal microscope.
matters needing attention
① After loading the probe, be sure to wash away any remaining probes that have not entered the cell, otherwise it will cause a higher background.
② After the probe is loaded and residual probes are cleaned, excitation wavelength scanning and emission wavelength scanning can be performed to confirm the loading status of the probe
Good.
③ Try to shorten the time from probe loading to measurement (excluding stimulation time) as much as possible to reduce various possible errors.
④ Based on the overall fluorescence intensity displayed in the results, the dilution factor and incubation time of the DCFH-DA probe can be adjusted appropriately.
Back
