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ROS Brite™ APF *Optimized for Detecting Reactive Oxygen Species (ROS)*

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Physical properties
Molecular weight423.42
Spectral properties
Excitation (nm)498
Emission (nm)517
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Excitation (nm)
Emission (nm)
Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen. Examples include superoxide, hydroxyl radical, singlet oxygen and peroxides. ROS is highly reactive due to the presence of unpaired valence shell electrons. ROS forms as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis. However, during times of environmental stress (e.g., UV or heat exposure), ROS levels can increase dramatically. This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. ROS are also generated by exogenous sources such as ionizing radiation. 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 with a variety of pathological events, including atherosclerosis, carcinogenesis, ischemic reperfusion injury, and neurodegenerative disorders. ROS Brite™ APF is a fluorogenic probe to measure hydroxyly radical in cells using conventional fluorescence microscopy, high-content imaging, microplate fluorometry, or flow cytometry. The cell-permeant ROS Brite™ APF reagent is nonfluorescent and produces bright green fluorescence upon reaction with hydroxyl radical. The resulting fluorescence can be measured using fluorescence imaging, high-content imaging, microplate fluorometry, or flow cytometry. In the presence of peroxidase, APF also reacts with hydrogen peroxide. APF has good selectivity to hydroxyl radical compared to other ROS. APF and HPF show relatively high resistance to light-induced oxidation. APF and HPF are nonfluorescent until they react with the hydroxyl radical or peroxynitrite anion. APF will also react with the hypochlorite anion.


Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)FITC filter set

Example protocol


Catalog Number

ROS Brite™ Dyes

Molecular Weight




ROS Brite™ APF


490 nm

515 nm


ROS Brite™ HPF


490 nm

515 nm


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. ROS Brite™ APF stock solution:
Prepare a 10 to 20 mM ROS Brite™ APF stock solution in DMSO. Note: The stock solution can be stored at -20C in single use aliquotes. Protect from light.


ROS Brite™ APF working Solution:
Make 1 to 10 µM working solution by diluting the DMSO stock solution into Hanks solution with 20 mM Hepes buffer (HHBS). Note: The working solution should be made fresh before use.


  1. Incubate the cells with ROS Brite™ APF (1-10 µM) for 20 - 60 minutes at 37°C.

  2. Replace the dye-loading solution with HHBS buffer.

  3. Analyze the cells with a proper fluorescence instrument at Ex/Em = 490/525 mm (cut off = 515 nM) with bottom read mode (e.g., FITC filter set for a fluorescence microscope, FL1 filter for a flow cytometer). Note: BSA and phenol red can affect the fluorescence and should be used with caution. APF can be used in solution assays or for intracellular measurements.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of ROS Brite™ APF *Optimized for Detecting Reactive Oxygen Species (ROS)* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM236.172 µL1.181 mL2.362 mL11.809 mL23.617 mL
5 mM47.234 µL236.172 µL472.344 µL2.362 mL4.723 mL
10 mM23.617 µL118.086 µL236.172 µL1.181 mL2.362 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles


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Spectral properties

Excitation (nm)498
Emission (nm)517



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View all 22 references: Citation Explorer
Developmental toxicity evaluation of three hexabromocyclododecane diastereoisomers on zebrafish embryos
Authors: Du M, Zhang D, Yan C, Zhang X.
Journal: Aquat Toxicol (2012): 1
MAPK inhibitors and siRNAs differentially affect cell death and ROS levels in arsenic trioxide-treated human pulmonary fibroblast cells
Authors: Park WH., undefined
Journal: Oncol Rep (2012): 1611
MG132, a proteasome inhibitor, induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion
Authors: Park WH, Kim SH.
Journal: Oncol Rep (2012): 1284
Enhancement of gallic acid-induced human pulmonary fibroblast cell death by N-acetyl cysteine and L-buthionine sulfoximine
Authors: You BR, Park WH.
Journal: Hum Exp Toxicol (2011): 992
Proteasome inhibition by MG132 induces growth inhibition and death of human pulmonary fibroblast cells in a caspase-independent manner
Authors: You BR, Park WH.
Journal: Oncol Rep (2011): 1705
MAPK inhibitors differentially affect gallic acid-induced human pulmonary fibroblast cell growth inhibition
Authors: Park WH., undefined
Journal: Mol Med Report (2011): 193
Gallic acid-induced lung cancer cell death is accompanied by ROS increase and glutathione depletion
Authors: You BR, Kim SZ, Kim SH, Park WH.
Journal: Mol Cell Biochem (2011): 295
beta-Lapachone induces heart morphogenetic and functional defects by promoting the death of erythrocytes and the endocardium in zebrafish embryos
Authors: Wu YT, Lin CY, Tsai MY, Chen YH, Lu YF, Huang CJ, Cheng CM, Hwang SP.
Journal: J Biomed Sci (2011): 70
Mitogen-activated protein kinase inhibitors differently affect the growth inhibition and death of a proteasome inhibitor, MG132-treated human pulmonary fibroblast cells
Authors: Park WH., undefined
Journal: Hum Exp Toxicol (2011): 1945
Hydrogen protects vestibular hair cells from free radicals
Authors: Taura A, Kikkawa YS, Nakagawa T, Ito J.
Journal: Acta Otolaryngol Suppl (2010): 95