Reactive oxygen species assay kit is a kit for reactive oxygen species detection using fluorescent probe DCFH-DA. DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by intracellular esterase to generate DCFH. DCFH, on the other hand, cannot penetrate the cell membrane, making it easy for probes to be loaded into cells. Intracellular reactive oxygen species can oxidize non-fluorescent DCFH to generate fluorescent DCF. Detection of DCF fluorescence can know the level of intracellular reactive oxygen species. This kit provides the reactive oxygen species positive control reagent Rosup to facilitate the detection of reactive oxygen species. Rosup is a compound mixture at a concentration of 50mg/ml.
1. Matters needing attention
1. After the probe is loaded, be sure to wash the remaining probes that have not entered the cells, otherwise the background will be high.
2. After the probe is loaded and the residual probe is cleaned, the scanning of the excitation wavelength and the scanning of the emission wavelength can be performed to confirm whether the loading of the probe is in good condition.
3. Try to shorten the time from probe loading to measurement (except stimulation time) to reduce various possible errors.
4. For your safety and health, please wear a lab coat and disposable gloves for operation.
2. Instructions for use
1. Load the probe
For cells with a short stimulation time (usually within 2 hours), load the probe first, and then stimulate the cells with reactive oxygen species positive controls and drugs of interest. For cells with long cell stimulation time (usually more than 6 hours), first stimulate cells with reactive oxygen species positive control and drugs of interest, and then load probes.
In situ loading of probes: This method is only applicable to adherent cultured cells. DCFH-DA was diluted 1:1000 with serum-free medium to a final concentration of 10 μmol/L. Remove the cell culture medium and add an appropriate volume of diluted DCFH-DA. The volume to be added should be sufficient to cover the cells. Usually, the volume of diluted DCFH-DA added to one well of a six-well plate is 1 ml. Incubate in a 37°C cell incubator for 20 minutes. Cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells.
Usually ROS positive controls can significantly increase ROS levels after stimulating cells for 20-30 minutes.
Probe loading after cell collection: DCFH-DA was diluted 1:1000 with serum-free medium to a final concentration of 10 μmol/L. After the cells were collected, they were suspended in diluted DCFH-DA at a cell concentration of 1 million to 20 million/ml, and incubated in a 37°C cell incubator for 20 minutes. Mix by inversion every 3-5 minutes to allow adequate contact between the probe and the cells. Cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells. Directly stimulate the cells with reactive oxygen species positive control and drugs of interest, or divide the cells into several parts and stimulate the cells. Usually ROS positive controls can significantly increase ROS levels after stimulating cells for 20-30 minutes.
2. Detection
Samples loaded with probes in situ can be directly observed with a laser confocal microscope, or cells can be collected and detected with a fluorescence spectrophotometer, a fluorescence microplate reader, or a flow cytometer.
Samples loaded with probes after collecting cells can be detected by fluorescence spectrophotometer, fluorescence microplate reader or flow cytometer, or directly observed by laser confocal microscope.
3. Parameter setting
Using the excitation wavelength of 488 nm and the emission wavelength of 525 nm, the intensity of fluorescence before and after stimulation was detected in real time or by time point. The fluorescence spectrum of DCF is very similar to that of FITC, and DCF can be detected with the parameter settings of FITC. The excitation and emission spectra of DCF refer to the figure below.
4. Other instructions
Positive controls can be used at a ratio of 1:1000. For example, a total of 1 ml of cells loaded with probes can be stimulated by adding 1 microliter of positive control. A very significant increase in reactive oxygen species levels is usually observed within 20-30 minutes of stimulation. For different cells, the effect of reactive oxygen species positive control may be quite different. If no increase in reactive oxygen species is observed within 30 minutes after stimulation, the concentration of reactive oxygen species positive control can be appropriately increased. If the active oxygen rises too fast, the concentration of the active oxygen positive control can be appropriately reduced.
In addition, for some cells, if the negative control cells without stimulation are found to have strong fluorescence, DCFH-DA can be diluted 1:2000-1:5000, so that the concentration of DCFH-DA when the probe is loaded is 2-5 micromolar /Lift. The probe loading time can also be adjusted appropriately within 15-60 minutes according to the situation.
