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 fluorescent 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 detection CellROX® Green Reagent, for oxidative stress detection CM-H2DCFDA, general oxidative stress indicator
Target Reactive oxygen Reactive oxygen Reactive oxygen
Reporter CellROX® Deep Red CellROX® Green CM-H2DCFDA
Ex/Em (nm) 644/665 485/520 495/525
Live cell Yes Yes Yes
Lysate Yes Yes Yes
Purified enzyme Yes Yes Yes
Usage Cell permeant for live-cell determinations Cell permeant for live-cell determinations Cell permeant for live-cell determinations
Components Bulk reagent Bulk reagent Bulk reagent
Format 5 x 50 µL 5 x 50 µL 20 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. C10422 C10444 C6827
  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)
Target Nitric oxide Nitric oxide Glutathione
Reporter DAF-FM DAF-FM ThiolTracker™ Violet
Ex/Em (nm) 495/515 495/515 405/526
Live cell No Yes Yes
Lysate Yes Yes Yes
Purified enzyme Yes Yes Yes
Usage Lower detection limit ~3 nM of NO Lower detection limit ~3 nM of NO Cell permeant for live-cell determinations
Components Bulk reagent Bulk reagent Bulk reagent
Format 1 mg 1 mg 500 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. D23841 D23842 T10096
  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)
Target Hydrogen peroxide/Peroxidase Use singly for general ROS detection; in tandem can be used to detect hypochlorous acid (HOCl) Targeted to mitochondria Blue fluorescent dye becomes red when oxidized
Reporter Amplex® Red reagent ARF HPF MitoSOX® Red Dihydroethidium (DHE)
Ex/Em (nm) 571/585
490/515
510/580 518/606
Live cell No
Yes
Yes Yes
Lysate Yes
No
No No
Purified enzyme Yes
No
NO No
Usage Lower 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 anions Detects increases in cellular superoxide production Exhibits blue-fluorescence in the cytosol until oxidized, where it intercalates within the cell's DNA
Components Includes assay buffers and controls Bulk reagent Bulk reagent Bulk reagent Bulk reagent
Format 500 assays using 100 µL reaction volume 470 µL 470 µL 10 x 50 µg 10 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. A22188 A36003 H36004 M36008 D11347
  EnzChek® Myeloperoxidase (MPO) Activity Assay Kit
Target Continuously detects chlorination and peroxidation activity of myeloperoxidase (MPO)
Reporter Amplex® UltraRed
Ex/Em (nm) 571/585
Live cell No
Lysate Yes
Purified enzyme Yes
Usage Measure 1.5 to 200 ng/mL MPO
Components Includes assay buffers and controls
Format 200 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
For Research Use Only. Not for use in diagnostic procedures.