Measurement of Reactive oxygen species (ROS) in Blood using DCFDA dye ?

10 February 2023 1 4K Report

I want to measure reactive oxygen species in whole blood. Can anyone please suggest a protocol or method for it using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dye ?

Geir Bjorklund

The 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dye can be used to measure reactive oxygen species (ROS) in whole blood. Here is a protocol you can follow:

Obtain fresh whole blood and add DCFDA dye to the blood sample at a final concentration of 10 μM.

Incubate the blood sample with DCFDA at 37°C for 30 minutes in the dark to allow the dye to penetrate the cells.

After incubation, immediately analyze the fluorescence intensity of the sample using a fluorescence spectrophotometer or a flow cytometer. The fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm will correspond to the level of ROS in the sample.

To confirm that the fluorescence signal is specific for ROS, you can perform a control experiment by incubating the blood sample with DCFDA in the presence of a ROS scavenger, such as N-acetylcysteine.

To obtain quantitative data, you can compare the fluorescence intensity of your sample to that of a standard curve created using known concentrations of hydrogen peroxide.

It is important to note that the protocol described above may need to be modified based on the specific requirements of your experiment and the type of analysis you are performing.

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