Dcfh-da (dichlorodihydrofluorescein-acetoacetate) itself has no fluorescence, can freely enter the cell through the living cell membrane, and is hydrolyzed by intracellular esterase to form DCFH. DCFH has no fluorescence and cannot penetrate the cell membrane, and is oxidized by intracellular reactive oxygen species to produce fluorescent DCF. According to the generation of fluorescence in living cells, cells can determine the content of active oxygen and change. It can be directly observed by flow cytometry or fluorescence microscope. It is a classical method for the detection of reactive oxygen species in tissues or living cells. Rosup positie for reactive oxygen species induced by drugs, according to the fluorescence signal intensity, the real level of reactive oxygen species can be.

1, the positive control Rosup is generally used at a concentration of 100μ M (recommended concentration 100-400μ M, depending on the cell type). A significant increase in reactive oxygen species levels was usually observed 30 min-4 h after stimulation. For different cells, the effect of active oxygen positive control may have bigger difference. If no increase in ROS is observed within 30 min after stimulation, the induction time can be prolonged or the concentration of ROS positive control can be appropriately increased. If the ROS level increases too rapidly, the induction time can be shortened or the concentration of the ROS positive control can be appropriately reduced.

2, experiment process, if it finds no stimulus negative control cells, fluorescence is strong, can according to the 1:2000-00 1:50 dilution DCFH - DA, make DCFH - DA final concentration of 2-5 & mu; M. The probe loading time can also be appropriately adjusted within 15-60 min depending on the situation.

3, Reactive oxygen species positive control (Rosup) is only used as a positive control of the sample, not in every sample to add reactive oxygen species positive control.

4, probe after loading, be sure to wash the residual not enter the probe of cells, otherwise it will lead to higher background.