Microplate Assays for Reactive Oxygen Species

Generation 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.

Generalized reactive oxygen assays

CellROX Reagents are fluorogenic probes for measuring generalized oxidative stress in cells. The dyes are nonfluorescent in a reduced state and fluoresce bright green, orange, or deep red upon oxidation. Assays are simple and reliable with a sensitive and robust readout, and the reagents can be applied to cells in complete growth medium.

Nitric oxide assays

DAF-FM and DAF-FM diacetate can be used to detect and quantify low concentrations of nitric oxide (NO). The reagents are essentially nonfluorescent until reacted with NO to form a fluorescent benzotriazole. DAF-FM fluorescence can be detected by any fluorescence microplate reader that can detect fluorescein.

Selective reactive oxygen indicators

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. Hydrogen peroxide reacts with nucleic acids, proteins, and lipids and can result in cell and tissue damage.

Myeloperoxidase (MPO)

Amplex UltraRed reagent (in the EnzChek MPO Activity Assay Kit) provides rapid and sensitive determination of both chlorination and peroxidation activities of MPO in solution and in cell lysates. The mix-and-read, room temperature assay can be used for continuous detection of these activities, making it ideal for high-throughput strategies.

DAF-FM
Fluorescence emission spectra of DAF-FM in solutions containing zero to 1.2 μM nitric oxide (NO) radical.

Selection guides
 CellROX Deep Red Reagent, for oxidative stress detectionCellROX Green Reagent, for oxidative stress detectionCM-H2DCFDA, general oxidative stress indicator
TargetReactive oxygenReactive oxygenReactive oxygen
ReporterCellROX Deep Red CellROX GreenCM-H2DCFDA
Ex/Em (nm)644/665485/520495/525
Live cellYesYesYes
LysateYesYesYes
Purified enzymeYesYesYes
UsageCell permeant for live-cell determinationsCell permeant for live-cell determinationsCell permeant for live-cell determinations
ComponentsBulk reagentBulk reagentBulk reagent
Format5 x 50 µL5 x 50 µL20 x 50 µg
Protocol outline
  1. Treat cells.
  2. Add CellROX reagent.
  3. Incubate 30 min at 37°C.
  4. Remove and wash 3X.
  5. Analyze cells.
  1. Treat cells.
  2. Add CellROX reagent.
  3. Incubate 30 min at 37°C.
  4. Remove and wash 3X.
  5. Analyze cells.
  1. Load cells with CM-H2DCFDA.
  2. Treat cells.
  3. Incubate 30 min at 37°C.
  4. Analyze cells.
Cat. No.C10422C10444C6827
 DAF-FM (4-Amino-5-Methylamino-2′,7′-Difluorofluorescein)DAF-FM Diacetate (4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate)ThiolTracker Violet (Glutathione Detection Reagent)
TargetNitric oxideNitric oxideGlutathione
ReporterDAF-FMDAF-FMThiolTracker Violet
Ex/Em (nm)495/515495/515405/526
Live cellNoYesYes
LysateYesYesYes
Purified enzymeYesYesYes
UsageLower detection limit ~3 nM of NOLower detection limit ~3 nM of NOCell permeant for live-cell determinations
ComponentsBulk reagentBulk reagentBulk reagent
Format1 mg1 mg500 assays using 100 µL reaction volume
Protocol outline
  1. Place cells in wells.
  2. Add solution to be tested.
  3. Incubate, then remove solution.
  4. Add DAF-FM diacetate.
  5. Incubate.
  6. Measure fluorescence.
  1. Prepare dye working solution.
  2. After drug treatment, add dye to cells.
  3. Incubate 30 min at 37°C.
  4. Analyze cells.
Cat. No.D23841D23842T10096
 Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (500 assays)APF (hydroxyl radical, hypochlorite or peroxynitrite sensor) HPF (hydroxyl radical and peroxynitrite sensor) MitoSOX Red Mitochondrial Superoxide Indicator, for live-cell imaging
Dihydroethidium (hydroethidine)
TargetHydrogen peroxide/PeroxidaseUse singly for general ROS detection; in tandem can be used to detect hypochlorous acid (HOCl)Targeted to mitochondriaBlue fluorescent dye becomes red when oxidized
ReporterAmplex Red reagentARFHPFMitoSOX RedDihydroethidium (DHE)
Ex/Em (nm)571/585
490/515
510/580518/606
Live cellNo
Yes
YesYes
LysateYes
No
NoNo
Purified enzymeYes
No
NONo
UsageLower detection limit 1 x 10–5 U/ml HRP Both reagents become fluorescent on reaction with ROS–APF exhibits higher response than HPF to hypochlorite anionsDetects increases in cellular superoxide productionExhibits blue-fluorescence in the cytosol until oxidized, where it intercalates within the cell's DNA
ComponentsIncludes assay buffers and controlsBulk reagentBulk reagentBulk reagentBulk reagent
Format500 assays using 100 µL reaction volume470 µL470 µL10 x 50 µg10 x 1 mg
Protocol outline
  1. Prepare standard curve samples and load into wells.
  2. Dilute experimental samples into reaction buffer and load into wells.
  3. Prepare the Amplex Red reagent working solution.
  4. Add Amplex Red solution to wells.
  5. Incubate.
  6. Measure fluorescence.
  1. Prepare reagent and add to cells.
  2. Incubate 20–60 min at 4–37°C depending on cell type.
  3. Wash to remove excess probe.
  4. Measure fluorescence.
  1. Prepare reagent and add to cells.
  2. Incubate 20–60 min at 4–37°C depending on cell type.
  3. Wash to remove excess probe.
  4. Measure fluorescence.
  1. Prepare reagent and add to cells.
  2. Incubate 10 min at 37°C, protect from light.
  3. Wash to remove excess probe.
  4. Measure fluorescence.
  1. Prepare reagent and add to cells.
  2. Incubate 20–60 min at 4–37°C depending on cell type.
  3. Wash to remove excess probe.
  4. Measure fluorescence.
Cat. No.A22188A36003H36004M36008D11347
 EnzChek Myeloperoxidase (MPO) Activity Assay Kit
TargetContinuously detects chlorination and peroxidation activity of myeloperoxidase (MPO)
ReporterAmplex UltraRed
Ex/Em (nm)571/585
Live cellNo
LysateYes
Purified enzymeYes
UsageMeasure 1.5 to 200 ng/mL MPO
ComponentsIncludes assay buffers and controls
Format200 assays using 100 µL reaction volume
Protocol outline
  1. Add sample or MPO standard into wells.
  2. Add diluted Amplex UltraRed reagent.
  3. Incubate for 30 min (room temp).
  4. Measure fluorescence intensity.
  5. Subtract background from sample and control.
  6. Generate MPO standard curve, determine sample concentration.
Cat. No.E33856
Generalized ROS assays
 CellROX Deep Red Reagent, for oxidative stress detectionCellROX Green Reagent, for oxidative stress detectionCM-H2DCFDA, general oxidative stress indicator
TargetReactive oxygenReactive oxygenReactive oxygen
ReporterCellROX Deep Red CellROX GreenCM-H2DCFDA
Ex/Em (nm)644/665485/520495/525
Live cellYesYesYes
LysateYesYesYes
Purified enzymeYesYesYes
UsageCell permeant for live-cell determinationsCell permeant for live-cell determinationsCell permeant for live-cell determinations
ComponentsBulk reagentBulk reagentBulk reagent
Format5 x 50 µL5 x 50 µL20 x 50 µg
Protocol outline
  1. Treat cells.
  2. Add CellROX reagent.
  3. Incubate 30 min at 37°C.
  4. Remove and wash 3X.
  5. Analyze cells.
  1. Treat cells.
  2. Add CellROX reagent.
  3. Incubate 30 min at 37°C.
  4. Remove and wash 3X.
  5. Analyze cells.
  1. Load cells with CM-H2DCFDA.
  2. Treat cells.
  3. Incubate 30 min at 37°C.
  4. Analyze cells.
Cat. No.C10422C10444C6827
Nitric oxide assays
 DAF-FM (4-Amino-5-Methylamino-2′,7′-Difluorofluorescein)DAF-FM Diacetate (4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate)ThiolTracker Violet (Glutathione Detection Reagent)
TargetNitric oxideNitric oxideGlutathione
ReporterDAF-FMDAF-FMThiolTracker Violet
Ex/Em (nm)495/515495/515405/526
Live cellNoYesYes
LysateYesYesYes
Purified enzymeYesYesYes
UsageLower detection limit ~3 nM of NOLower detection limit ~3 nM of NOCell permeant for live-cell determinations
ComponentsBulk reagentBulk reagentBulk reagent
Format1 mg1 mg500 assays using 100 µL reaction volume
Protocol outline
  1. Place cells in wells.
  2. Add solution to be tested.
  3. Incubate, then remove solution.
  4. Add DAF-FM diacetate.
  5. Incubate.
  6. Measure fluorescence.
  1. Prepare dye working solution.
  2. After drug treatment, add dye to cells.
  3. Incubate 30 min at 37°C.
  4. Analyze cells.
Cat. No.D23841D23842T10096
Selective ROS indicators
 Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (500 assays)APF (hydroxyl radical, hypochlorite or peroxynitrite sensor) HPF (hydroxyl radical and peroxynitrite sensor) MitoSOX Red Mitochondrial Superoxide Indicator, for live-cell imaging
Dihydroethidium (hydroethidine)
TargetHydrogen peroxide/PeroxidaseUse singly for general ROS detection; in tandem can be used to detect hypochlorous acid (HOCl)Targeted to mitochondriaBlue fluorescent dye becomes red when oxidized
ReporterAmplex Red reagentARFHPFMitoSOX RedDihydroethidium (DHE)
Ex/Em (nm)571/585
490/515
510/580518/606
Live cellNo
Yes
YesYes
LysateYes
No
NoNo
Purified enzymeYes
No
NONo
UsageLower detection limit 1 x 10–5 U/ml HRP Both reagents become fluorescent on reaction with ROS–APF exhibits higher response than HPF to hypochlorite anionsDetects increases in cellular superoxide productionExhibits blue-fluorescence in the cytosol until oxidized, where it intercalates within the cell's DNA
ComponentsIncludes assay buffers and controlsBulk reagentBulk reagentBulk reagentBulk reagent
Format500 assays using 100 µL reaction volume470 µL470 µL10 x 50 µg10 x 1 mg
Protocol outline
  1. Prepare standard curve samples and load into wells.
  2. Dilute experimental samples into reaction buffer and load into wells.
  3. Prepare the Amplex Red reagent working solution.
  4. Add Amplex Red solution to wells.
  5. Incubate.
  6. Measure fluorescence.
  1. Prepare reagent and add to cells.
  2. Incubate 20–60 min at 4–37°C depending on cell type.
  3. Wash to remove excess probe.
  4. Measure fluorescence.
  1. Prepare reagent and add to cells.
  2. Incubate 20–60 min at 4–37°C depending on cell type.
  3. Wash to remove excess probe.
  4. Measure fluorescence.
  1. Prepare reagent and add to cells.
  2. Incubate 10 min at 37°C, protect from light.
  3. Wash to remove excess probe.
  4. Measure fluorescence.
  1. Prepare reagent and add to cells.
  2. Incubate 20–60 min at 4–37°C depending on cell type.
  3. Wash to remove excess probe.
  4. Measure fluorescence.
Cat. No.A22188A36003H36004M36008D11347
Myeloperoxidase (MPO)
 EnzChek Myeloperoxidase (MPO) Activity Assay Kit
TargetContinuously detects chlorination and peroxidation activity of myeloperoxidase (MPO)
ReporterAmplex UltraRed
Ex/Em (nm)571/585
Live cellNo
LysateYes
Purified enzymeYes
UsageMeasure 1.5 to 200 ng/mL MPO
ComponentsIncludes assay buffers and controls
Format200 assays using 100 µL reaction volume
Protocol outline
  1. Add sample or MPO standard into wells.
  2. Add diluted Amplex UltraRed reagent.
  3. Incubate for 30 min (room temp).
  4. Measure fluorescence intensity.
  5. Subtract background from sample and control.
  6. Generate MPO standard curve, determine sample concentration.
Cat. No.E33856

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