This method can be used to analyze cellular reactive oxygen species in adherent cells with only a fluorescence microscope, and quantify reactive oxygen species intensity with fluorescence plate reader. This protocol is simple, efficient, and cost-effective. Start by seeding two times 10 to the fifth HCT116 colorectal cancer cells in DMEM into each well of a 24-well plate.
Incubate the cells overnight at 37 degrees Celsius. On the next day, replace the culture medium with 100 micromolar ferrous sulfate, or 10 micromolar doxorubicin-containing medium, and incubate the plate for another 24 hours. Prepare the DCFH-DA solution by dissolving 4.85 milligrams of DCFH-DA in one milliliter of DMSO to make a 10 millimolar stock solution.
Right before adding it to the wells, dilute the stock with pre-warmed DMEM to make a 10 micromolar working solution, and vortex it for 10 seconds. 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 times G for 10 minutes at four 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 one 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. HCT116 colorectal cancer cells were treated with 100 micromolar ferrous sulfate, or 10 micromolar doxorubicin, to induce oxidative stress. As expected, green fluorescence was dramatically increased by both treatments.
To quantify the relative intensity changes, the cells were lysed and normalized with protein concentrations. The relative fluorescence intensity was significantly increased by ferrous sulfate or doxorubicin. When performing this procedure, it is important to make DCFH-DA solution fresh and avoid light exposure, as well as to minimize cell status disturbance, and to perform extensive PBS wash right before taking image.
In addition to detection of the total ROS by this protocol, specifically ROS such as peroxide can be performed.
