A Novel Fluorescent Probe for Imaging and Detecting Hydroxyl Radical in Living Cells

by Jinfang Liao, Jixiang Liu, Qin Zhao, Yunting Xi, Zhenjun Diwu

Introduction

The hydroxyl radical (•OH) is the most reactive oxygen species (ROS), and plays a significant role in a variety of diseases. In general, hydroxyl radical is considered to be a harmful by-product of oxidative metabolism, which can cause molecular damage in a living system. Therefore, sensitive and selective detection of intracellular hydroxyl radical is essential to understand cellular redox and the impact of its dysregulation on various pathologies. Although a variety of fluorescent sensors have been developed to detect hydroxyl radicals, their rapid photobleaching, short emission wavelengths and non-selective reactions with other ROS species have limited their applications in cells and tissues. To address this unmet need, we have developed a novel fluorescent probe to selectively detect intracellular hydroxyl radical in living cells.

The novel hydroxyl radical probe (MitoROS™ OH580) is live-cell permeant and can rapidly generate red fluorescence when it reacts with •OH in cells. In the absence of •OH, the probe has no significant absorption and negligible fluorescence. This fluorogenic characteristic makes this new probe have high signal-to-noise ratio. The newly developed fluorescent probe also has high selectivity for •OH over other reactive oxygen species (ROS) and reactive nitrogen species (RNS), enabling the specific detection in a biological system. We anticipate that MitoROS™ OH580 can serve as a valuable tool for life science research and medical diagnostic applications.

Materials and Methods

Cell Culture

HeLa and Raw 264.7 cells were seeded overnight in 30,000 to 100,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate at 37°C.

Hydroxyl Radical Generation

HeLa cells were treated with Fenton reaction (10 µM CuCl₂ and 100 µM H₂O₂) and 37°C for 1 hour to induce exogenous hydroxyl radical.
RAW 264.7 cells were treated with PMA (phorbol 12-myristate 13-acetate) in growth medium at 37°C for 4 hours to stimulate endogenous hydroxyl radical.

Hydroxyl Radical Assay

Hydroxyl radical assay: Cells were stained with MitoROS™ OH580 at 37°C for 1 hour prior to treatment. Wash cells 2-3 times before fluorescence imaging.

Imaging Hydroxyl Radical in Living Cells

Fig. 1

Imaging exogenous hydroxyl radical (•OH) in RAW 264.7 macrophage cells. Red: hydroxyl radical stained with MitosROS™ OH580 (Cat No.16055); Blue: cell nuclei stained with Hoechst 33342 (Cat No.17530).

Fig. 2

Imaging endogenous hydroxyl radical (•OH) in RAW 264.7 macrophage cells. Red: hydroxyl radical stained with MitoROS OH580 (Cat#16055); Green: cell nuclei stained with Nuclear Green LCS1 (Cat#17540).

Representative Data of ROS Reagents in Living Cells

Fig. 3

Imaging superoxide (O₂^•-) in HeLa cells (Cat No.16060).

Representative Data of RNS Reagents in Living Cells

Fig. 4

Flow data Cat No. 16351;Microplate data Cat No. 16315

A: Flow cytometry data of detecting nitric oxide (NO) in Jurkat cells (Cat No.16351).B: Plate reader data of detecting peroxynitrite (ONOO-) in RAW 264.7 cells (Cat No.16315).

Probes for imaging ROS and RNS species in living cells

Intracellular ROS detection reagents and assays.

Selective ROS detection reagents and assays.

Document: 02.0017.151117r1

Last updated Thu Oct 23 2025