Oxidative Stress DetectionGeneration of reactive oxygen species (ROS) is inevitable for aerobic organisms and, in healthy cells, occurs at a controlled rate. Under conditions of oxidative stress, ROS production is dramatically increased, resulting in subsequent alteration of membrane lipids, proteins, and nucleic acids. Oxidative damage of these biomolecules is associated with aging as well as a variety of pathological events, including atherosclerosis, carcinogenesis, ischemia reperfusion injury, and neurodegenerative disorders.

 

 

Life Technologies provides a variety of Molecular Probes® fluorescent tools to track different parameters in oxidative stress:

These tools use platforms such as fluorescence microscopy, flow cytometry, or microplate analysis.

Select products for oxidative stress detection

 

 

Oxidative stress detection video

Detection of oxidative stress with CellROX® Green in U2OS cells.

Generalized oxidative stress

CellROX® Reagents are fluorogenic probes for measuring generalized oxidative stress in cells using conventional fluorescence microscopy, high content screening, microplate fluorometry, or flow cytometry. The dyes are nonfluorescent in a reduced state and fluoresce bright green, orange, or deep red upon oxidation. Some are formalin fixable for easy multiplexing with other fluorescent reagents including antibodies. Assays are simple and reliable with a sensitive and robust readout, and the reagent can be applied to cells in complete growth media. Life Technologies provides CellROX® dyes and conventional probes for oxidative stress measurement.

 Oxidative stress detection with CellROX® Green Reagent
General oxidative stress detection in U2OS cells with CellROX® Green Reagent.

Lipid peroxidation

Lipid peroxidation is the oxidative degradation of lipids. ROS are the major initiators of lipid peroxidation, and membrane-bound polyunsaturated fatty acids like arachidonic acid and linoleic acid are their major targets. The byproducts of lipid peroxidation cause direct damage to cell membranes. They also form protein adducts, resulting in cell and tissue damage. Lipid peroxidation is implicated in many human diseases including diabetes and cardiovascular disease. Life Technologies provides Molecular Probes® tools for live and fixed cells, with a choice of ratiometric or single-wavelength readout.

  Detection of lipid peroxidation with the Image-iT® Lipid Peroxidation Kit
Detection of lipid peroxidation with the Image-iT® Lipid Peroxidation Kit.

Selective ROS detection

Superoxide, peroxyl radical, hydrogen peroxide, hydroxyl radical, and peroxynitrite are examples of ROS that react with nucleic acids, proteins, and lipids and result in cell and tissue damage. Certain ROS have been implicated in various human diseases including cancer, cardiovascular disease, neurodegenerative disease, and aging. Life Technologies provides selective Molecular Probes® reagents for a variety of reactive species targeted to the cytosol or mitochondria and detected using imaging, flow cytometry, or microplate analysis.

  Detection of ROS with MitoSOX™ Red Superoxide Indicator
Detection of superoxide in live cells using MitoSOX™ Red Superoxide Indicator.

Fluorescent protein–based redox sensors

Genetically encoded sensors provide an alternative to organic dyes for measuring redox potential or oxidative stress in live cells. roGFP is a fluorescent protein chimera that changes its excitation maximum from 488 nm to 400 nm based on a highly efficient redox relay. Using ratiometric detection of the emission at 515 nm, this shift can be used to measure H2O2 (Premo™ Cellular Hydrogen Peroxide H2O2 Sensor) or redox potential (Premo™ Cellular Redox Sensor, Grx-1-roGFP)  in live cells in a reversible and reproducible assay. The fluorescent proteins can be multiplexed with organic dyes or other expressed proteins to provide additional biological context for the assay.

  Premo™ Cellular Redox Sensor
Detecting changes in cellular redox potential in U2OS cells using Premo™ Cellular Redox Sensor

Glutathione

Reduced glutathione, also known as GSH, is a major thiol bound to proteins. Protein thiols including GSH play an important role in determining the redox status of cells. Therefore, detection of GSH levels is a useful indication of redox potential and a cell’s ability to prevent oxidative stress. Life Technologies provides Molecular Probes® intracellular probes for the sensitive detection and localization of thiols.

  Detection of glutathione with ThiolTracker™ Violet dye
Detection of GSH in U2OS cells with ThiolTracker™ Violet dye.

Selection Guides for Oxidative Stress Detection Products

Oxidative stress results from an imbalance in the production of reactive oxygen species (ROS) and the ability of the cell to scavenge them. ROS react with nucleic acids, proteins and lipids causing cell and tissue damage and can be measured using selective or general indicators.

 
Readout CellROX® Dyes are non-fluorescent in a reduced state and exhibit bright, stable fluorescence upon oxidation by reactive oxygen species (ROS). They are membrane permeant and load readily into live cells Non-fluorescent dye becomes green when oxidized
Common filter set Cy®5 RFP FITC FITC
Reporter CellROX® Deep Red Reagent CellROX® Orange Reagent CellROX® Green Reagent H2-DCFDA
Ex/Em (nm) 640/665 545/565 485/520 495/527
Live cell–compatible Yes Yes Yes Yes
Labeling in complete medium Yes Yes Yes No
Formaldehyde-fixable Yes No Yes No
Detergent-resistant No No Yes No
Photostability
Signal-to-noise ratio
Bibliography Citations    Citations
Imaging Yes Yes Yes Yes
HCS Yes Yes Yes Yes
Microplate Yes Yes Yes Yes
Flow cytometry C10491 (kit) C10493 (kit) C10492 (kit) Yes
Format 5 x 50 uL 5 x 50 uL 5 x 50 uL 20 x 50 ug
Cat. No. C10422 C10443 C10444 C6827

Lipid peroxidation is the oxidative degradation of lipids. Reactive oxygen species are the major initiators of lipid peroxidation and membrane bound polyunsaturated fatty acids like arachidonic acid and linoleic acid are their major targets. The byproducts of lipid peroxidation cause direct damage to cell membranes. They also form protein adducts resulting in cell and tissue damage. Lipid peroxidation is implicated in many human diseases including diabetes and cardiovascular disease.

 
Image-iT® Lipid Peroxidation Kit,
for live cell analysis
Readout Ratiometric indicator of lipid peroxidation shifts from red to green fluorescence with oxidation Single wavelength indicator detects lipid-peroxidation derived protein modifications in fixed cells
Common filter set(s) FITC and Texas Red® FITC
Fluorophore BODIPY® 581/591 C11 Alexa Fluor® 488
Ex/Em (nm)
495/519 581/591 495/519
Live cell–compatible Yes No
Detects fixed cells No Yes
Whole cell imaging Yes Yes
Live cell–compatible Yes No
Photostability
Signal-to-noise ratio
Bibliography Citations Citations
Formaldehyde-fixable No Detects fixed cells
Antibody-multiplexable/
Detergent-resistant
No Yes
Imaging Yes Yes
HCS Yes Yes
Microplates Yes Yes
Flow cytometry Yes Yes
Format 500 assay kit 500 assay kit
Cat. No. C10445 C10446

Superoxide, peroxyl radical, hydrogen peroxide, hydroxyl radical and peroxynitrite are some examples of ROS that react with nucleic acids, proteins and lipids and result in cell and tissue damage. Certain ROS have been implicated in various human diseases including cancer, cardiovascular disease, neurodegenerative disease and aging.

  Dihydroethidium Premo™ Cellular Hydrogen Peroxide (H2O2) Sensor
Specificity Superoxide (•O2) Superoxide (•O2) Nitric Oxide (NO) Hydrogen peroxide (H2O2)
Readout Targeted to mitochondria Blue fluorescent dye becomes red when oxidized Fluorescence increases with accumulation of NO Detects H2O2 levels with ratio of excitation at 400 and 488 nm and single emission at 515 nm
Common filter set Texas Red® RFP FITC None
Fluorophore MitoSOX™ Red Dihydroethidium (DHE) DAF-FM roGFP
Ex/Em (nm)
510/580 518/606 495/515 400 and 488/515
Live cells Yes Yes Yes Yes
Fixable No Yes No No
Labeling in complete medium No   No Yes
Photostability
Signal-to-noise ratio
Bibliography Citations Citations Citations  
Imaging Yes Yes Yes Yes
HCS Yes Yes Yes Yes
Microplate Yes   Yes Yes
Flow Cytometry Yes   Yes Yes
In vivo     Yes No
Format 10 x 50 ug 10 x 1 mg 10 x 50 ug 5 mL
Cat. No. M36008 D11347 D23844 P36243

Reduced glutathione also known as GSH is a major thiol bound to proteins. Protein thiols including GSH play an important role in determining the redox status of cells. Therefore, detection of reduced GSH levels is a useful indication of redox potential and a cell's ability to prevent oxidative stress.

 
Readout Intracellular probe for GSH used in subcellular detection and localization as well as monitoring of sub populations Reacts with several low molecular weight thiols to generate fluorescent conjugates Detects redox potential with ratio of excitation wavelength and single emission
Common filter set DAPI longpass DAPI None
Ex/Em (nm)
404/525 394/490 400 and 488/515
Fluorophore ThiolTracker™ Violet Monochlorobimane (mBCI) Monobromobimane (mBBr) roGFP
Live cell–
compatible
Yes Yes Yes
Formaldehyde-fixable Yes No No
Antibody-multiplexable Yes No No
Whole cell imaging Yes Yes Yes
Photostability
Signal-to-noise
ratio
Bibliography Citations Citations Citations  
Imaging Yes   Yes   Yes
HCS Yes   Yes   Yes
Microplates   Yes Yes Yes Yes
Flow cytometry Yes   Yes Yes Yes
Format 180 assay kit 500 assay kit 25 mg 25 mg 5 mL
Cat. No. T10095 T10096 M1381MP M1378 P36424