ROS Detection with DCFH-DA Staining: Measuring Total ROS in Colorectal Cancer Cell Lines

- Reactive oxygen species are produced predominantly from mitochondria when electrons leak out from the electron transport chain. These electrons combine with molecular oxygen giving rise to various reactive oxygen species, or ROS. In cancer cells, there is overproduction of reactive oxygen species. These highly reactive ROS molecules can oxidatively damage membranes, proteins, and DNA inside the cell.

Increased levels of ROS damages DNA leading to activation of an oncogenic signaling pathway causing cancer progression. Therefore, it is vital to detect the levels of reactive oxygen species produced by the cancer cells.

Begin by taking cancer cells from human colorectal cancer in an appropriate culture media. Next, seed these cells to a multi-well plate and add DCFH-DA solution. Incubate the plate for the desired duration. DCFH-DA crosses the cell membrane. Once inside the cell, DCFH-DA converts to DCFH in the presence of a cellular esterase enzyme. Further, the oxidation of DCFH by reactive oxygen species yields green fluorescent molecules.

Observe the cells under a fluorescent microscope. The intensity of green fluorescence indicates the level of reactive oxygen species in the cell. In the following protocol, we use DCFH-DA dye to stain reactive oxygen species in the colorectal cancer cells.

- To stain the cells, remove the drug-containing medium and wash them once with DMEM. Add 500 microliters of DCFH-DA working solution into each well and incubate the plate at 37 degrees Celsius for 30 minutes. After the incubation, remove the DCFH-DA from the wells and wash them twice with DMEM and once with PBS.

Then, add 500 microliters of PBS to each well. Use the green fluorescent protein channel on the fluorescence microscope to take representative images of the cells. Then, remove the PBS and add 200 microliters of radioimmunoprecipitation assay buffer to each well.

Incubate the plate on ice for five minutes and collect the cell lysates into 1.5 milliliter tubes. Centrifuge the tubes at 21,130 x g for 10 minutes at 4 degrees Celsius. Then, transfer 100 microliters of the supernatant to a black 96-well plate. Use a microplate reader at an excitation wavelength of 485 nanometers and an emission wavelength of 530 nanometers to measure the fluorescence in each well.

Next, measure the protein concentration with the Bradford assay by transferring 1 microliter of the supernatant to a clear 96-well plate with 100 microliters of protein assay solution. Use the protein concentrations to normalize the fluorescence intensities.