Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Summary

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

Abstract

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Introduction

Three major reactive oxygen species (ROS) produced by cellular metabolism that are of physiological meaning are superoxide anion, hydroxyl radical, and hydrogen peroxide¹. At low concentrations, they participate in physiological cell processes, but at high concentrations they have adverse effects on cell signaling pathways¹. Our body has developed antioxidant systems, which are effective against excessive ROS. However, oxidative stress can occur when ROS overwhelm the detoxifying ability of our body, which contributes to many pathological conditions, including inflammation, cancer, and neurodegenerative disease^2,3,4. The purpose of this method is to determine total cellular ROS in adherent cells using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The rationale is that oxidation of DCFH-DA to 2’-7’dichlorofluorescein (DCF) has been used extensively for total ROS detection including hydroxyl radicals (•OH) and nitrogen dioxide (•NO₂). Mechanistically, DCFH-DA is taken up by cells where cellular esterase cleaves off the acetyl groups, resulting in DCFH. Oxidation of DCFH by ROS converts the molecule to DCF, which emits green fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Compared with detection of fluorescence with flow cytometry and other alternative methods⁵, advantages of this method using a fluorescence microscope and a plate reader are that it produces clearly visible fluorescent images, and is easy to perform, efficient and cost-effective. This method has been widely used to detect cellular ROS for studying various conditions^6,7,8. This protocol is used for detecting total ROS in adherent cells. Using this method to detect ROS in suspension cells may need some modifications.

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 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 a 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 assay⁹.
7. Normalize fluorescence intensities with protein concentrations.

Results

HCT116 colorectal cancer cells were treated with 100 µM FS or 10 µM DOX to induce oxidative stress⁷. As shown in Figure 1, green fluorescence was dramatically increased by both FS and DOX as expected. To quantify the relative intensity change, the cells were lysed after taking images and normalized with protein concentrations. The quantified fluorescence intensity was significantly increased by FS or DOX in HCT116 cells.

Discussion

The experimental protocol described here is easily reproducible to measure cellular total ROS. The critical steps include making DCFH-DA solution fresh and avoiding light exposure, minimizing cell status disturbance and extensive PBS washing right before taking images. For the preparation of DCFH-DA working solution, the stock solution should be added into pre-warmed DMEM right before adding into the 24 well plate. The reason is that old solutions that generate high background fluorescence or light exposure will lead to ...

Materials

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
Ferrous Sulfate Heptahydrate	VWR, Radnor, PA	97061-542
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