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

Introduction

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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 radical, their rapid photo-bleaching, short emission wavelength and non-selective reaction with other ROS species have limited their applications in cell 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

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Cell Culture

1. 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

1. 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.
2. 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

1. 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

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Representative Data of ROS Reagents in Living Cells

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Representative Data of RNS Reagents in Living Cells

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Probes for imaging ROS and RNS species in living cells

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Intracellular ROS detection reagents and assays.

ROS Species	ROS Brite™ 570	ROS Brite™ 670	ROS Brite™ 700	ROS Brite™ HDCF	Amplite® ROS Green	Amplite® ROS Red
Hydrogen Peroxide (H2O2)	+	+	+	+++	+++	+++
Hydroxyl radical (•OH)	++	++	++	+	+	+
Tert-butyl-hyrdoperoxide (TBHP)	+	+	+	+	+	+
Hypochlorous acid (HOCl)	-	+	++	-	+	-
Superoxide anion (•O2-)	+	++	++	-	-	-
Nitric Oxide (NO)	-	-	-	-	-	-
Peroxynitrite anion (ONOO-)	-	-	-	-	-	-
Cat No.	22902	22903	16004	16053	22900 22904	22901

Selective ROS detection reagents and assays.

ROS Species	OxiVision™ Green	OxiVision™ Blue	MitoROS™ 520	MitoROS™ 580	MitoROS™ OH580
Hydrogen Peroxide (H2O2)	+++	+++	-	-	-
Hydroxyl radical (•OH)	-	-	-	-	+++
Tert-butyl-hyrdoperoxide (TBHP)	-	-	-	-	-
Hypochlorous acid (HOCl)	-	-	-	-	-
Superoxide anion (•O2-)	-	-	+++	+++	-
Nitric Oxide (NO)	-	-	-	-	-
Peroxynitrite anion (ONOO-)	-	-	-	-	-
Cat No.	11503 11506	11504 11505	16060	22970 22971	16055