High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Summary

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

Abstract

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

Introduction

Reactive oxygen species (ROS) are a group of naturally occurring, highly reactive, and temporally unstable chemical radicals formed as a part of the normal cellular metabolism in cells. ROS plays a key and essential role in the modulation of normal physiological and biochemical processes occurring in cells^1,2. The main source of ROS production in cells is from the mitochondrial electron transport chain (ETC) pathway as a part of the normal bioenergetic cycle. Significant additional sources of ROS production include enzymatic reactions such as cellular NADPH oxidases in cells. Metabolism of food molecules ....

Protocol

1. Cell culture

1. Seed the test cells (HepG2, HUH7, and JHH4 hepatocellular carcinoma cells) into a 96-well plate at a seeding density of 10,000 cells/well in a final seeding volume of 200 µL per well.
   1. Before culturing the HepG2 cells, coat the 96 plate wells with type IV collagen (50 µg/mL) for 2 h duration at room temperature (RT). To avoid solidification of the stock collagen, place the stock solution into ice and subsequently, initiate the dilution process to the desir.......

Discussion

In this study, a protocol to assess superoxide-driven intracellular reactive oxygen species (ROS) production using dihydroethidium (DHE) fluorescence dye was established on a high-content screening system. A majority of the current protocols available in the literature use the DCFH-DA as a fluorescence imaging probe to quantify ROS species. However, multiple studies have shown the DCFH-DA is not an ideal probe for the measurement of intracellular ROS. Various reasons postulated for the unsuitability of DCFH-DA as a probe.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL centrifuge tubes	VWR	20170-038
96- well plate	Corning Costar	07-200-90
Cellomics Cx7	ThermoFisher	HCSDCX7LEDPRO
Collagen	Advanced Biomatrix	5056
DHE (Dihydroethidium)	ThermoFisher	D1168
DMEM	Sigma	6046
FBS	VWR	97068-085
GraphPad Prism	GraphPad	Version 6.0
HepG2 cell line	ATCC
Hoechst	ThermoFisher	33342
HUH7 cell line	ATCC
Hydrogen Peroxide	Sigma	88597
JHH4 cell line	ATCC
Menadione	Sigma	M5625