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.

 

 

We provide a variety of Invitrogen 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

 

Detection of oxidative stress with Invitrogen™ CellROX™ Green Reagent in U2OS cells.

Generalized oxidative stress

Invitrogen 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. We provide Invitrogen 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. We provide Invitrogen 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 Invitrogen 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. We provide selective Invitrogen 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 Invitrogen 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 (Invitrogen Premo Cellular Hydrogen Peroxide H2O2 Sensor) or redox potential (Invitrogen 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. We provide Invitrogen intracellular probes for the sensitive detection and localization of thiols.

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

 
ReadoutCellROX 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 cellsNon-fluorescent dye becomes green when oxidized
Common filter setCy5RFPFITCFITC
ReporterCellROX Deep Red ReagentCellROX Orange ReagentCellROX Green ReagentH2-DCFDA
Ex/Em (nm)640/665545/565485/520495/527
Live cell–compatibleYesYesYesYes
Labeling in complete mediumYesYesYesNo
Formaldehyde-fixableYesNoYesNo
Detergent-resistantNoNoYesNo
Photostability
Signal-to-noise ratio
BibliographyCitations   Citations
ImagingYesYesYesYes
HCSYesYesYesYes
MicroplateYesYesYesYes
Flow cytometryC10491 (kit)C10493 (kit)C10492 (kit)Yes
Format5 x 50 uL5 x 50 uL5 x 50 uL20 x 50 ug
Cat. No.C10422C10443C10444C6827

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
ReadoutRatiometric indicator of lipid peroxidation shifts from red to green fluorescence with oxidationSingle wavelength indicator detects lipid-peroxidation derived protein modifications in fixed cells
Common filter set(s)FITC and Texas RedFITC
FluorophoreBODIPY 581/591 C11Alexa Fluor 488
Ex/Em (nm)
495/519581/591495/519
Live cell–compatibleYesNo
Detects fixed cellsNoYes
Whole cell imagingYesYes
Live cell–compatibleYesNo
Photostability
Signal-to-noise ratio
BibliographyCitations Citations
Formaldehyde-fixableNoDetects fixed cells
Antibody-multiplexable/
Detergent-resistant
NoYes
ImagingYesYes
HCSYesYes
MicroplatesYesYes
Flow cytometryYesYes
Format500 assay kit500 assay kit
Cat. No.C10445C10446

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.

 DihydroethidiumPremo Cellular Hydrogen Peroxide (H2O2) Sensor
SpecificitySuperoxide (•O2)Superoxide (•O2)Nitric Oxide (NO)Hydrogen peroxide (H2O2)
ReadoutTargeted to mitochondriaBlue fluorescent dye becomes red when oxidizedFluorescence increases with accumulation of NODetects H2O2 levels with ratio of excitation at 400 and 488 nm and single emission at 515 nm
Common filter setTexas RedRFP FITCNone
FluorophoreMitoSOX RedDihydroethidium (DHE)DAF-FMroGFP
Ex/Em (nm)
510/580518/606495/515400 and 488/515
Live cellsYesYesYesYes
FixableNoYesNoNo
Labeling in complete mediumNo NoYes
Photostability
Signal-to-noise ratio
BibliographyCitations CitationsCitations  
ImagingYesYesYesYes
HCSYesYesYesYes
MicroplateYes YesYes
Flow CytometryYes YesYes
In vivo  YesNo
Format10 x 50 ug10 x 1 mg10 x 50 ug5 mL
Cat. No.M36008D11347D23844P36243

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.

 
ReadoutIntracellular probe for GSH used in subcellular detection and localization as well as monitoring of sub populationsReacts with several low molecular weight thiols to generate fluorescent conjugatesDetects redox potential with ratio of excitation wavelength and single emission
Common filter setDAPI longpassDAPINone
Ex/Em (nm)
404/525394/490400 and 488/515
FluorophoreThiolTracker VioletMonochlorobimane (mBCI)Monobromobimane (mBBr)roGFP
Live cell–
compatible
YesYesYes
Formaldehyde-fixableYesNoNo
Antibody-multiplexableYesNoNo
Whole cell imagingYesYesYes
Photostability
Signal-to-noise
ratio
BibliographyCitations Citations Citations 
ImagingYes Yes Yes
HCSYes Yes Yes
Microplates YesYesYesYes
Flow cytometryYes YesYesYes
Format180 assay kit500 assay kit25 mg25 mg5 mL
Cat. No.T10095T10096M1381MPM1378P36424

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.

 
ReadoutCellROX 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 cellsNon-fluorescent dye becomes green when oxidized
Common filter setCy5RFPFITCFITC
ReporterCellROX Deep Red ReagentCellROX Orange ReagentCellROX Green ReagentH2-DCFDA
Ex/Em (nm)640/665545/565485/520495/527
Live cell–compatibleYesYesYesYes
Labeling in complete mediumYesYesYesNo
Formaldehyde-fixableYesNoYesNo
Detergent-resistantNoNoYesNo
Photostability
Signal-to-noise ratio
BibliographyCitations   Citations
ImagingYesYesYesYes
HCSYesYesYesYes
MicroplateYesYesYesYes
Flow cytometryC10491 (kit)C10493 (kit)C10492 (kit)Yes
Format5 x 50 uL5 x 50 uL5 x 50 uL20 x 50 ug
Cat. No.C10422C10443C10444C6827

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
ReadoutRatiometric indicator of lipid peroxidation shifts from red to green fluorescence with oxidationSingle wavelength indicator detects lipid-peroxidation derived protein modifications in fixed cells
Common filter set(s)FITC and Texas RedFITC
FluorophoreBODIPY 581/591 C11Alexa Fluor 488
Ex/Em (nm)
495/519581/591495/519
Live cell–compatibleYesNo
Detects fixed cellsNoYes
Whole cell imagingYesYes
Live cell–compatibleYesNo
Photostability
Signal-to-noise ratio
BibliographyCitations Citations
Formaldehyde-fixableNoDetects fixed cells
Antibody-multiplexable/
Detergent-resistant
NoYes
ImagingYesYes
HCSYesYes
MicroplatesYesYes
Flow cytometryYesYes
Format500 assay kit500 assay kit
Cat. No.C10445C10446

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.

 DihydroethidiumPremo Cellular Hydrogen Peroxide (H2O2) Sensor
SpecificitySuperoxide (•O2)Superoxide (•O2)Nitric Oxide (NO)Hydrogen peroxide (H2O2)
ReadoutTargeted to mitochondriaBlue fluorescent dye becomes red when oxidizedFluorescence increases with accumulation of NODetects H2O2 levels with ratio of excitation at 400 and 488 nm and single emission at 515 nm
Common filter setTexas RedRFP FITCNone
FluorophoreMitoSOX RedDihydroethidium (DHE)DAF-FMroGFP
Ex/Em (nm)
510/580518/606495/515400 and 488/515
Live cellsYesYesYesYes
FixableNoYesNoNo
Labeling in complete mediumNo NoYes
Photostability
Signal-to-noise ratio
BibliographyCitations CitationsCitations  
ImagingYesYesYesYes
HCSYesYesYesYes
MicroplateYes YesYes
Flow CytometryYes YesYes
In vivo  YesNo
Format10 x 50 ug10 x 1 mg10 x 50 ug5 mL
Cat. No.M36008D11347D23844P36243

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.

 
ReadoutIntracellular probe for GSH used in subcellular detection and localization as well as monitoring of sub populationsReacts with several low molecular weight thiols to generate fluorescent conjugatesDetects redox potential with ratio of excitation wavelength and single emission
Common filter setDAPI longpassDAPINone
Ex/Em (nm)
404/525394/490400 and 488/515
FluorophoreThiolTracker VioletMonochlorobimane (mBCI)Monobromobimane (mBBr)roGFP
Live cell–
compatible
YesYesYes
Formaldehyde-fixableYesNoNo
Antibody-multiplexableYesNoNo
Whole cell imagingYesYesYes
Photostability
Signal-to-noise
ratio
BibliographyCitations Citations Citations 
ImagingYes Yes Yes
HCSYes Yes Yes
Microplates YesYesYesYes
Flow cytometryYes YesYesYes
Format180 assay kit500 assay kit25 mg25 mg5 mL
Cat. No.T10095T10096M1381MPM1378P36424