AAT Bioquest

Detection of Reactive Oxygen Species (ROS) in Live Cell Mitochondria

by Jinfang Liao, Ruogu Peng, Yunting Xi, Zhen Luo, Zhenjun Diwu, Jixiang Liu, Qin Zhao


Mitochondrial oxidative stress has received considerable attention in recent years not only as the primary source of intracellular reactive oxygen species (ROS) production but also as a major contributor to public health-related diseases, such as asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, various neurodegenerative diseases and Down's syndrome. However, many of the available reagents for detecting mitochondrial ROS species either lack the sensitivity needed for targeting live cell mitochondria or the selectivity to measure individual ROS.

At AAT Bioquest, we have developed a novel series of mitochondrial ROS fluorescence probes that not only localize specifically in live cell mitochondria, but also can selectively detect the different ROS species, including superoxide, hydroxyl radical and hydrogen peroxide in multiple Ex/Em wavelengths.

Cancer cells stained with our novel fluorescence probes showed negligible fluorescence in absence of ROS stimulants. In contrast, OxiVision™ Blue-loaded cells with hydrogen peroxide displayed strong blue fluorescence in mitochondria, while MitoROS OH580-loaded cells with Fenton reaction (to induce hydroxyl radical) showed strong red fluorescence in mitochondria and MitoROS™ 520-loaded cells exhibited strong green fluorescence in mitochondria after treated with superoxide stimulants. In addition, all four probes have been successfully used in flow cytometry, fluorescence microscopy and fluorescence-based micorplate assays to quantitatively detect mitocohndrial ROS in living cells.

In conclusion, these probes are mitochondrion-targeted ROS probes, which can be used for monitoring the exogenous and endogenous changes of ROS levels in living cells in real time by fluorescence imaging and flow cytometer.


Material and Methods

Cell Culture

  1. Cervical cancer HeLa cells and RAW 264.7 macrophage cells were seeded overnight in a 96-well black wall/clear bottom costar plate at 37 °C in DMEM. Non-adherent cell line Human T lymphocyte Jurkat cells were grown at 37 °C in RPMI 1640

Cell Treatment

  1. Dye-loaded cells were treated with hydrogen peroxide, Fenton reaction, PMA (phorbol 12-myristate 13-acetate), Antimycin A (AMA) and Pyo (Pyocyanin) to generate intracellular hydrogen peroxide (H2O2), exogenous & endogenous hydroxyl radical (•OH), and exogenous & endogenous superoxide (•O2-), respectively.


Table 1. Properties of mitochondrial ROS reagents.

ROS Probe
Target ROS/RNS Species
Ex (nm)
Em (nm)
Filter Set/Channel
OxiVision™ BlueMitochondrial H2O2405450DAPI
MitoROS™ 520Mitochondrial O2•-509534FITC
MitoROS™ 580Mitochondrial O2•-540590TRITC
MitoROS™ OH580Mitochondrial •OH576598TRITC


Mitochondrial Hydrogen Peroxide Detection Using OxiVision™ Blue

Fluorescence images

Fluorescence images of mitochondrial hydrogen peroxide (H2O2) in HeLa cells (A), and flow cytometric analysis of Jurkat cells (B) stained with OxiVision Blue for 30 minutes and treated with or without 100 µM H2O2 at 37 °C for 90 minutes. The fluorescence images were acquired using fluorescence microscope with a DAPI filter. Flow cytometric analysis were obtained using ACEA NovoCyte flow cytometer in Pacific Blue channel.


Imaging Mitochondrial Hydroxyl Radical Using MitoROS™ OH580

Fenton Reaction
MitoROS OH580 and Hoechst Co-stain
MitoROS OH580 and Hoechst co-stain_control

Fluorescence images of mitochondrial hydroxyl radical (•OH) in HeLa cells. Cells stained with MitoROS™ OH580 were treated with or without Fenton reaction (10 µM CuCl2 and 100 µM H2O2) at 37 °C for 1 hour to induce exogenous hydroxyl radical. Red: MitoROS™ OH580; Blue: Hoechst 33342.

PMA 100 ng/mL
MitoROS OH580 and Nuclear Green Co-stain_partA
PMA 10 ng/mL
MitoROS OH580 and Nuclear Green Co-stain_partB
MitoROS OH580 and Nuclear Green Co-stain_control

Fluorescence images of mitochondrial hydroxyl radical (•OH) in RAW 264.7 macrophage cells. Cells stained with MitoROS™ OH580 were treated with PMA (phorbol 12-myristate 13-acetate) in the range of 0-100 ng/mL in growth medium at 37 °C for 4 hours to stimulate endogenous hydroxyl radical. Red: MitoROS™ OH580; Green: Nuclear Green™ LCS1.


Mitochondrial Superoxide Detection Using MitoROS™ 520 and MitoROS™ 580

(A) MitoROS™ 520 AMA Treatment
MitoROS 520 AMA Treatment
(A) MitoROS™ 520 Control
MitoROS 520 Control

Treatment and Control

(B) MitoROS™ 580 AMA Treatment
MitoROS 580 AMA Treatment
(B) MitoROS™ 580 Control
MitoROS 580 Control

Treatment and Control

(C) Microplate Measurmements
(D) Flow Cytometric Analysis

Fluorescence images of mitochondrial superoxide (O2•-) in RAW 264.7 macrophage cells stained with (A) MitoROS™ 520 or (B) MitoROS™ 580 with or without 50 µM AMA (Antimycin A) treatment at 37 °C for 30 minutes. (C) HeLa cells seeded in a 96-well black wall/clear bottom plate, were stained with MitoROS™ 580 and incubated with 50 µM Pyo (Pyocyanin), 50 µM AMA or without treatment (Control) at 37 °C for 30 minutes. The signal were monitored at Ex/Em = 540/590 nm (cutoff = 570 nm) with bottom read mode using a CLARIOstar microplate reader (BMG Labtech). (D) Flow cytometric analysis of MitoROS™ 580 labeled Jurkat cells treated with or without 50 µM AMA at 37 °C for 1 hour. The fluorescence signal was monitored at FL2 channel using a flow cytometer (BD FACSCalibur).

Original created on July 7, 2020, last updated on July 7, 2020
Tagged under: mitochondria ROS, ROS Detection