2,7-Dichlorodihydrofluorescein (DCFH) is a non-fluorescent probe that is oxidized by intracellular reactive oxygen species (ROS) to produce the highly fluorescent 2',7'-Dichlorofluorescein (DCF).DCFH is commonly used to assay the intracellular level of ROS, to detect ROS production and to analyze oxidative stress.

Instructions
I. Solution preparation
1. Preparation of stock solution: Dissolve 4.85 mg 2,7-Dichlorodihydrofluorescein in 1 mL dimethyl sulfoxide (DMSO) to make a 10 mM stock solution.
2. Preparation of working solution: Dilute the stock solution to a 10 μM working solution with pre-warmed DMEM before adding it to the wells.
II. Operation steps
1. Cell seeding
Inoculate 2 × 10^5 HCT116 colorectal cancer cells per well in a 24-well plate and maintain the cells in Dulbecco's modified Eagle's medium (DMEM) at 37 °C overnight.
2. 2,7-Dichlorodihydrofluorescein staining
1) Remove the medium containing the drug and wash once with DMEM.
2) Add 500 μL of 2,7-Dichlorodihydrofluorescein working solution to each well and incubate at 37 °C for 30 minutes.
3) Remove the 2,7-Dichlorodihydrofluorescein working solution, wash once with DMEM and twice with 1× phosphate-buffered saline (PBS).
4) Add 500 μL of 1× PBS to each well.
3. Imaging acquisition and intensity measurement
1) Take representative fluorescence images for each well using the green fluorescent protein (GFP) channel on a fluorescence microscope.
2) After taking the image, remove the PBS and add 200 μL of radioimmunoprecipitation assay (RIPA) buffer to each well.
3) Incubate on ice for 5 minutes, then collect the cell lysate into a 1.5 mL tube.
4) Centrifuge at 21,130 × g for 10 minutes at 4 °C.
5) Transfer 100 μL of 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 supernatant to a clear 96-well plate containing 100 μL of 1× protein assay solution to measure protein concentration using Bradford assay.
7) Normalize the fluorescence intensity with the protein concentration.