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

1. Cell seeding

1. Seed 2 x 10⁵ HCT116 colorectal cancer cells per well in a 24-well plate and maintain the cells in Dulbecco's modified Eagle medium (DMEM) overnight at 37 °C.
2. Replace the culture medium with or without 100 µM ferrous sulfate (FS) or 10 µM doxorubicin (DOX) containing medium and incubate for 24 h.

2. Preparation of the DCFH-DA solution

1. Dissolve 4.85 mg of DCFH-DA in 1 mL of dimethyl sulfoxide (DMSO) to make a 10 mM stock solution.
2. Dilute the stock solution with pre-warmed DMEM into a 10 µM working solution right before adding it to the wells.
3. Vortex the working solution for 10 s.

3. DCFH-DA staining

1. Remove the drug-containing medium and wash once with DMEM.
2. Add 500 µL of the DCFH-DA working solution into each well and incubate at 37 °C for 30 min.
3. Remove the DCFH-DA working solution. Wash once with DMEM and 2x with 1x phosphate-buffered saline (PBS).
4. Add 500 µL of 1x PBS to each well.

4. Imaging acquisition and intensity measurement

1. Take representative fluorescent images for each well using the green fluorescent protein (GFP) channel on a fluorescence microscope.
2. After taking images, remove PBS and add 200 µL of radioimmunoprecipitation assay (RIPA) buffer to each well.
3. Incubate on ice for 5 min, then collect cell lysate into 1.5 mL tubes.
4. Centrifuge at 21,130 x g for 10 min at 4 °C.
5. Transfer 100 µL of the supernatant to a black 96-well plate and measure the fluorescence intensity using a fluorescence microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
6. Transfer 1 µL of the supernatant to a clear 96-well plate containing 100 µL of 1x protein assay solution to measure the protein concentration using the Bradford assay.
7. Normalize fluorescence intensities with protein concentrations.