1. Before opening the cover, please briefly centrifuge, and collect the liquid on the inner wall of the cover at the bottom of the tube to avoid the loss of the staining solution when opening the cover.

2, there should be a control without DHE incubation, that is, without probe, only cells resuspended in 0.01M PBS.

3, After labeling with fluorescent probes, it is important to wash any remaining probes that have not entered the cell to avoid background elevation. Pay attention to the operation standard when washing many times to avoid washing the cells off.

1. For cells with short drug treatment (incubation) time (within 2 hours, it is also recommended that within 4 hours), you can use fluorescent dye to mark the cells first, and then stimulate the cells with drugs. For cells treated with drugs for a long time (more than 4 hours or more than 6 hours), it is recommended to treat (stimulate) the cells with drugs first and then label them with the probe.

5, DHE is easy to be oxidized in light and air, so attention should be paid to avoid light during storage and operation.

6, for different cells and tissues, the appropriate incubation time and concentration should be selected to observe the changes in ROS.

7, for your safety and health, please wear a laboratory coat and wear disposable gloves to operate.

Equipment required

Flow cytometer, fluorescence microplate reader, laser confocal microscope, fluorescence spectrophotometer, etc.

Steps:

I. Cell samples:

1. Collection of cells for reactive oxygen species determination

(1) Cell collection: suspension cells: 2000 RPM, centrifugal 5 min, collect precipitation, with 0.01 M PBS serum-free culture or washing twice, 1000 RPM, centrifugal 5 min, abandon supernatant, take cells precipitation; Adherent cells: remove the culture medium by suction, blow with 0.01M PBS or serum-free culture medium repeatedly, make the cell layer all into PBS or culture medium, collect the cell suspension, wash with 0.01M PBS or serum-free culture medium twice, centrifuge at 1000rpm for 5min, discard the supernatant, take cell precipitation;

(2) Adding DHE probe: resuspend the cell precipitate with the diluted DHE fluorescent probe. In general, the cell density is required to be 1*106-2*107/ml, and the recommended initial working concentration of probe is 10μ Can be in 1 M (DHE work concentration & micro; M~100 μ In the range of M ~ 100 & micro, pre-experiment is needed to determine the optimal concentration).

(3) 37o C The cells were incubated for 10 min to 90 min. Typically, 10-30 minutes will do. Note: The length of incubation time depends on cell type, stimulation condition, DHE concentration, etc. It can be reversed and mixed every 5min so that the probe is in full contact with the sample.

(4) was centrifuged at 1000g for 5min, cell precipitates were collected by removing the supernatant, washing twice with PBS buffer, and cells were resuspended.

(5) was used for fluorescence detection, and the results were expressed as fluorescence value.

2, without collecting the cells, directly add the probe into the culture medium for determination

(1) Add the DHE probe: remove the cell culture medium supernatant, add the DHE probe diluted in serum-free medium (recommended final concentration of probe is 10μ M), the volume of the probe added should be suitable to cover the cells, usually no less than 500 µ per well in 6-well plate; l.

(2) 37o C The cells were incubated for 10 min to 90 min. Typically, 10-30 minutes will do. Note: The length of incubation time depends on cell type, stimulation condition, DHE concentration, etc. It can be reversed and mixed every 5min so that the probe is in full contact with the sample.

(3) Discard the upper culture medium and wash twice with 0.01M PBS or serum-free culture medium to remove the probe that has not entered the cells.

(4) was used for fluorescence detection, and the results were expressed as fluorescence value.

II. Animal tissue samples

1. Cell suspension preparation: Single cell suspension preparation instrument or traditional tissue processing methods such as enzymatic hydrolysis and grinding method can be used to prepare single cell suspension;

2, adding DHE probe, adding DHE probe: remove the cell culture medium supernatant, add the DHE probe diluted in serum-free culture medium (recommended final concentration of probe is 10μ M).

3, 37o C The cells were incubated for 10 min to 90 min. Typically, 10-30 minutes will do. Note: The length of incubation time depends on cell type, stimulation condition, DHE concentration, etc. It can be reversed and mixed every 5min so that the probe is in full contact with the sample.

4, 1000g, centrifugation for 5min, remove the supernatant to collect cell precipitation, wash twice with PBS buffer, resuspend the cells

5, fluorescence detection, the results are expressed as fluorescence value.