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.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

DMSO: 250 mg/mL (620.02 mM), Sonication is recommended.