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

Abstract

Source: Kim, H., et al. Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining. J. Vis. Exp. (2020).

This video describes the protocol for detecting total cellular reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). This method helps visualize ROS localization in the adherent cells using a fluorescence microscope and further quantify ROS intensity with a fluorescence plate reader.

Protocol

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 soluti....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2',7'-Dichlorofluorescein diacetate	Cayman Chemical, Ann Arbor, MI	20656
Doxorubicin hydrochloride	TCI America, Portland, OR	D4193-25MG
Dulbecco's Modified Eagle Medium	Corning, Corning, NY	45000-304
Invitrogen EVOS FL Auto Imaging System	Thermo Fisher Scientific Waltham, MA	AMAFD1000	or any other fluorescence microscope
Protein assay Bradford solution	Bio-Rad, Hercules, CA	5000001
SpectraMax M2 Microplate Reader	Molecular Devices, Radnor, PA	89429-532	or any other fluorescence microplate reader
Ferrous Sulfate Heptahydrate	VWR, Radnor, PA	97061-542