Shop All Reactive Oxygen & Nitrogen Species

Fc OxyBURST™ Green Assay Reagent (Invitrogen™)

The Fc OxyBURST® Green assay reagent comprises bovine serum albumin (BSA) that has been covalently linked to dichlorodihydrofluorescein (H2DCF) and then complexed with a purified rabbit polyclonal anti-BSA antibody. When this immune complex binds to Fc receptors, the nonfluorescent H2DCF molecules are internalized within the phagovacuoloe and subsequently oxidized to green-fluorescent 2''7'-dichlorofluorescein.

APF (Hydroxyl Radical, Hypochlorite or Peroxynitrite Sensor) (Invitrogen™)

Aminophenyl fluorescein (APF) and hydroxyphenyl fluorescein (HPF, H36004), two reactive oxygen species (ROS) indicators developed by T. Nagano, offer greater specificity and stability than dichlorodihydrofluorescein diacetate (H2DCFDA, D399). APF and HPF show limited non-selective reactivity and relatively high resistance to light-induced oxidation. Both of these new fluorescein derivatives are nonfluorescent until they react with the hydroxyl radical or peroxynitrite anion; APF will also react with the hypochlorite anion. Together, these two ROS indicators can selectively detect hypochlorite anion. Upon oxidation, both APF and HPF exhibit bright green fluorescence (excitation/ emission maxima ~490/515 nm), making them compatible with any instrument that can detect fluorescein.

Griess Reagent Kit, for nitrite quantitation (Invitrogen™)

The Greiss reagent kit provides a simple and well characterized colorimetric assay for nitrites, and nitrates that have been reduced to nitrites, with a detection limit of about 100 nM.

CellROX™ Green Reagent, for oxidative stress detection (Invitrogen™)

CellROX® Green Reagent is a novel fluorogenic probe for measuring oxidative stress in live cells. The cell-permeant dye is weakly fluorescent while in a reduced state and exhibits bright green photostable fluorescence upon oxidation by reactive oxygen species (ROS) and subsequent binding to DNA, with absorption/emission maxima of ~ 485/520 nm. This reagent can be formaldehyde-fixed and its signal survives detergent treatment, which makes it multiplexable with other compatible dyes and antibodies.

CellROX® Green Reagent is compatible with various platforms, such as traditional fluorescence microscopy, high content screening (HCS), flow cytometery, and microplate-based fluorimetry or high throughput screening (HTS). This reagent is also compatible with various benchtop instruments such as our FLoid™, Tali™, and Attune™ instruments.

In addition, CellROX™ Green Reagent is:

• Compatible with other live cell dyes, antibodies, and in cells expressing RFP
• Provided as a stable, ready-to-use DMSO solution with a simple no-wash protocol compatible with standard workflows in fluorescence microscopy
• Provided as five single-use vials containing sufficient total reagent for five 96-well plates or 100 coverslips

Oxidative stress results from an imbalance between the production of reactive oxygen species (ROS) and the ability of cells to scavenge them. ROS play an important role in the progression of several diseases, including inflammation, atherosclerosis, aging, and age-related degenerative disorders.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

BODIPY™ 665/676 (Lipid Peroxidation Sensor) (Invitrogen™)

Lipid peroxidation can be detected with the lipophilic probe, BODIPY® 665/676 dye. This probe exhibits a change in fluorescence after interaction with peroxyl radicals.

CellROX™ Deep Red Reagent, for oxidative stress detection (Invitrogen™)

CellROX® Deep Red Reagent is a novel fluorogenic probe for measuring cellular oxidative stress in both live and fixed cell imaging, with absorption/emission maxima at ~644/665 nm. The cell-permeant dye is non-fluorescent while in a reduced state, and exhibits bright fluorescence upon oxidation by reactive oxygen species (ROS).

Features of the CellROX® Deep Red Reagent:

• Optimized probe for detection of oxidative stress in cells
• Simple protocol that can be used with other live cell dyes and GFP
• Compatible with both live cell fluorescence-imaging and formaldehyde-based fixation methods

Substrate Specifics
The CellROX® Deep Red reagent is a novel cell-permeant dye with absorption/emission maxima of ~644/665 nm. CellROX® Deep Red reagent is non-fluorescent while in a reduced state and becomes fluorescent upon oxidation by reactive oxygen species with emission maxima ~665 nm that is measurable by fluorescent imaging, high content imaging, fluorescent plate readers, or flow cytometry.

Oxidative Stress Detection with CellROX® Deep Red Reagent
Oxidative stress results from an imbalance between production of reactive oxygen species (ROS) and the ability of cells to scavenge them. ROS play an important role in the progression of several diseases including inflammation, atherosclerosis, aging, and age-related degenerative disorders. CellROX® Deep Red Reagent can detect the oxidative stress in cells by reacting with ROS to become brightly fluorescent.

Simple, Robust Protocol
The protocol to use this reagent is simple and the bright deep red fluorescence signal is compatible with other live cell dyes and GFP, making it useful in multiplex fluorescence assays to measure a variety of cellular phenomena, including parameters related to cytotoxicity and cell death (Figure 4 below). Furthermore, unlike many other ROS sensors, such as H2DCFDA, the signal from CellROX® Deep Red is retained after formaldehyde fixation for flexibility with the assay and improved workflows compared to those based on classic dyes for ROS detection.

Image-iT™ Lipid Peroxidation Kit, for live cell analysis (Invitrogen™)

The Image-iT® Lipid Peroxidation Kit enables the detection of lipid peroxidation in live cells through oxidation of BODIPY® 581/591 C11 reagent. This reagent localizes to membranes throughout live cells and upon oxidation by lipid hydroperoxides, displays a shift in peak fluorescence emission from ~590 nm to ~510 nm. Fluorescence from live cells shifts from red to green, providing a ratiometric indication of lipid peroxidation compatible with traditional and high content microscopy, as well as flow cytometry. This kit also includes cumene hydroperoxide as a positive control compound to induce lipid peroxidation in cells.

Additional Information:

• The reagent is provided as a stable, ready-to-use DMSO solution with a simple protocol compatible with standard workflows in fluorescence microscopy
• The reagent is provided as five single-use vials containing sufficient total reagent for five 96-well plates or 100 coverslips

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

BODIPY™ 581/591 C11 (Lipid Peroxidation Sensor) (Invitrogen™)

BODIPY® 581/591 undecanoic acid can be used to detect reactive oxygen species (ROS) in cells and membranes. Oxidation of the polyunsaturated butadienyl portion of the dye results in a shift of the fluorescence emission peak from ~590 nm to ~510 nm.

RedoxSensor™ Red CC-1 - Special Packaging (Invitrogen™)

RedoxSensor Red CC-1 is a unique probe whose fluorescence localization appears to be based on a cell's cytosolic redox potential. Upon passively entering live cells, the stain is either oxidized in the cytosol to a red-fluorescent product (excitation/emission maxima ~540/600 nm), which then accumulates in mitochondria, or the probe is transported to lysosomes, where it is subsequently oxidized. The differential distribution of the oxidized product appears to depend on the redox potential of the cytosol.

DAF-FM Diacetate (4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate) (Invitrogen™)

DAF-FM is a reagent that is used to detect and quantify low concentrations of nitric oxide (NO). It is essentially nonfluorescent until it reacts with NO to form a fluorescent benzotriazole. DAF-FM fluorescence can be detected by any instrument that can detect fluorescein, including flow cytometers, microscopes, fluorescent microplate readers and fluorometers.

DAF-FM Specifications:
• Ex/Em of DAF-FM: ~495/515 nm
• Lyophilized product should be dissolved using DMSO and then added to an aqueous buffer to create a working solution
• DAF-FM diacetate is cell permeant and passively diffuses across cellular membranes; once inside the cell, it is converted to a cell-impermeant form
• Buffers containing bovine serum albumin (BSA) and phenol red may affect the fluorescence and should be used with caution
• The fluorescence quantum yield of DAF-FM is ~0.005, but increases about 160-fold, to ~0.81, after reacting with NO


Applications of DAF-FM:
• Assessment of NO production in transaldolase-deficient lymphoblasts by flow cytometry
• Detection of NO accumulation in embryonic cortical neurons following neurotrophin stimulation
in vivo imaging of NO in zebrafish
• Intravital microscopic detection of NO generation associated with angiogenesis in mice
• Quantitation of ATP-induced NO release in rabbit platelets

Important Advantages of DAF-FM Over DAF-2
The spectra of the NO adduct of DAF-FM are independent of pH above pH 5.5. Also, the NO adduct of DAF-FM is significantly more photostable than that of DAF-2, which means additional time for image capture. Finally, DAF-FM is a more sensitive reagent for NO than is DAF-2 (NO detection limit for DAF-FM ~3 nM versus ~5 nM for DAF-2).

Probes for Nitric Oxide Research
We offer several products for the detection of reactive oxygen species (ROS) and nitric oxide (NO). Review these products in Probes for Nitric Oxide Research—Section 18.3 in the Molecular Probes® Handbook.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

Di(Acetoxymethyl Ester) (6-Carboxy-2',7'-Dichlorodihydrofluorescein Diacetate) (Invitrogen™)

6-carboxy-2',7'-dichlorodihydrofluorescein diacetate is a chemically reduced, acetylated form of fluorescein used as an indicator for reactive oxygen species (ROS) in cells. This nonfluorescent molecule is readily converted to a green-fluorescent form when the acetate groups are removed by intracellular esterases and oxidation (by the activity of ROS) occurs within the cell. We offer the carboxylated H2DCFDA analog (carboxy-H2DCFDA, C400), which has two negative charges at physiological pH, and its di(acetoxymethyl ester) (C2938). Upon cleavage of the acetate and ester groups by intracellular esterases and oxidation, both analogs form carboxydichlorofluorescein (C368), with additional negative charges that impede its leakage out of the cell.

ROS Indicator Specifications:
• Ex/Em: ~492–495/517–527 nm
• Product is air sensitive and should be stored under dry argon or nitrogen
• Product may be dissolved in DMSO, DMF, or ethanol for use
• Indicator is cell permeant (cell loading protocols are available in the literature)
• Fluorescence can be monitored using a flow cytometer, fluorometer, microplate reader, or fluorescence microscope, using excitation sources and filters appropriate for fluorescein


Find More ROS Indicators
We offer an assortment of Molecular Probes® products for the generation of reactive oxygen species (ROS), including singlet oxygen, superoxide, hydroxyl radical and various peroxide and hydroperoxides, as well as for their fluorometric detection in solution. Review Generating and Detecting Reactive Oxygen Species—Section 18.2 in the Molecular Probes® Handbook for more information on these products.

For Research Use. Not for human or animal therapeutic or diagnostic use.

Click-iT™ Lipid Peroxidation Imaging Kit - Alexa Fluor™ 488 (Invitrogen™)

The Click-iT® Lipid Peroxidation Imaging Kit - Alexa Fluor® 488 leverages copper-catalyzed click chemistry and the linoleamide alkyne (LAA) reagent (alkyne-modified linoleic acid) for detection of lipid peroxidation-derived protein modifications in fixed cells. Linoleic acid is the most abundant polyunsaturated fatty acid found in mammals and its lipid peroxidation products likely account for the majority of lipid-derived protein carbonyls.

The Click-iT® Lipid Peroxidation Imaging Kit is:
• Optimized for detection of lipid peroxidation in cells using click chemistry and Alexa Fluor® 488 azide
• Multiplexible and compatible with traditional fluorescence microscopy, high content screening (HCS), and flow cytometry
• Provided as a complete kit sufficient for five 96-well plates or 100 coverslips

How It Works
The Click-iT® Lipid Peroxidation Imaging Kit contains cumene hydroperoxide as a positive control for induction of lipid peroxidation and all of the components for the click reaction, including LAA, and Alexa Fluor® 488 azide. LAA can be incubated with cells, where it incorporates into cellular membranes. Upon lipid peroxidation, LAA is oxidized and produces 9- and 13- hydroperoxy-octadecadienoic acid (HPODE). These hydroperoxides decompose to multiple α,β-unsaturated aldehydes, which readily modify proteins at nucleophilic side chains. These alkyne-containing modified proteins can be subsequently detected using Click-iT® chemistry and multiplexed with other probes appropriate for fixed cells. This assay is amenable to detection by traditional fluorescence microscopy, high content screening (HCS), and flow cytometry.

Available in Two Formats
The Click-iT® Lipid Peroxidation Imaging Kit - Alexa Fluor ® 488 contains the components you need to detect lipid peroxidation in cells, including positive control compound. Alternatively, Click-iT® LAA (linoleamide alkyne) is a stand-alone reagent provided as a five pack set of single-use vials for maximum flexibility in assays for lipid peroxidation. This reagent may be combined with a variety of azide-modified detection reagents and other reagents in the Click-iT® tool box.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

Premo™ Cellular Redox Sensor (Invitrogen™)

Premo™ Cellular Redox Sensor is a genetically encoded sensor that is transduced/transfected into cells, enabling dynamic measurement of glutathione redox potential or oxidative stress in live cells. This detection system responds to stimuli quickly and is reversible, and can be readily combined with other dyes for multiplexed measurement of other relevant markers. Premo™ Cellular Redox Sensor can be used as an alternative method of detecting redox potential or oxidative stress, in concert with organic-based fluorescent dyes such as CellROX®, 2',7'-dichlorodihydrofluorescein diacetate (H2-DCFDA), or ThiolTrackers™ dyes. With the incorporated BacMam technology, this sensor is ready to use—simply add to cells in growth media and incubate overnight.

The roGFP-Grx1 Chimera
Premo™ Cellular Redox Sensor combines the selectivity of a roGFP-Grx1 chimera with the transduction efficiency of BacMam 2.0 technology. Redox-senstive green fluorescent protein (roGFP) detects reactive oxygen through two cysteines introduced into the β-barrel structure of the GFP protein. Dilsulfide formation between the cysteines leads to protonation of GFP and increases the 400 nm excitation spectra at the expense of the 488 nm excitation spectra. The fluorescence at the two different excitation wavelengths can be quantitated using the same emission wavelength of 515 nm. roGFP responds to levels of GSH/GSSG through electron exchange with glutaredoxin (GRX1). The availability of GRX1 to this dynamic reaction is ensured through the linking of GRX1 to roGFP, enabling roGFP to respond faster to stimulus and be more sensitive to GSH/GSSG with reversible functionality.

BacMam Technology
The incorporated BacMam technology is based on the use of an insect cell virus (baculovirus) to efficiently deliver and express genes in mammalian cells. Because mammalian cells do not support replication of baculoviruses, transduction is extremely well tolerated and generally lacking in cytopathic effects, even at high virus levels. Learn more about BacMam technology.

Resazurin, Sodium Salt (Invitrogen™)

The non-fluorescent resazurin can be used as a fluorogenic oxidation-reduction indicator in a variety of cells, including bacteria, yeast and eukaryotes by flow cytometry, fluorescence microscopy and high-throughput screening. The red-fluorescent product, resorufin exhibits absorption/emission maxima ~575/585 nm.

Glutathione Ethyl Ester, Biotin Amide (BioGEE) (Glutathiolation Detection Reagent) - Special Packaging (Invitrogen™)

Biotinylated glutathione ethyl ester (BioGEE) is a cell-permeant, biotinylated glutathione analog for the detection of glutathiolation. Under conditions of oxidative stress, cells may transiently incorporate glutathione into proteins. Stressed cells incubated in BioGEE will also incorporate this biotinylated glutathione derivative into proteins, facilitating the identification of oxidation-sensitive proteins. Once these cells are fixed and permeabilized, you can detect glutathiolation levels with a fluorescent streptavidin conjugate using either flow cytometry or fluorescence microscopy. Proteins glutathiolated with BioGEE can also be extracted and analyzed by mass spectrometry or by Western blotting methods in conjunction with fluorophore- or enzyme-labeled streptavidin conjugates.