Home>>Signaling Pathways>> Immunology/Inflammation>> Reactive Oxygen Species>>H2DCFDA (DCFH-DA)


Catalog No.: GC30006

H2DCFDA (DCFH-DA) (DCFH-DA) is a cell-permeable probe used to detect intracellular reactive oxygen species (ROS) (Ex/Em=488/525 nm).

H2DCFDA (DCFH-DA) Chemical Structure

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10mM (in 1mL DMSO)
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Sample solution is provided at 25 µL, 10mM.

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Cell experiment [1, 2]:

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Preparation Method

Telomerase-immortalized human corneal epithelial cells (hCECs) were cultured at 37℃ under 5% CO2 atmosphere in bronchial epithelium growth medium supplemented with 5 mg/mL insulin, 0.5 mg/mL hydrocortisone, a mixture of 50 mg/mL gentamicin and 50 ng/mL amphotericin, 5 ng/mL human epidermal growth factor, and 0.15 mg/mL BSA. They were then subcultured with 0.25% trypsin-EDTA every 3–4 days prior to use in this study. Incubate the cells with H2DCFDA(DCFH-DA). Then detached cells from the culture wells using 0.25% trypsin-EDTA and washed twice using ice-cold PBS. Flow cytometry measurements were performed three times for each treatment. .

Reaction Conditions

H2DCFDA (DCFH-DA) concentration:10 μM; incubate with hCECs at 37℃ for 30 minutes in the dark.


H2DCFDA(DCFH-DA) is a redox-sensitive fluorescent, which could be used to measure intracellular reactive oxygen species levels. It is normally deacetylated by cellular esterases into a non-fluorescent compound that is subsequently oxidized by ROS into 2′,7′-dichlorofluorescein (DCF). Then measure the DCF florescence at 485 and 535 nm of maximum excitation and emission spectra, respectively.


[1]. Park JH, Moon S-H, Kang DH, et al. Diquafosol sodium inhibits apoptosis and inflammation of corneal epithelial cells via activation of Erk1/2 and RSK: in vitro and in vivo dry eye model. Invest Ophthalmol Vis Sci. 2018; 59:5108–5115. doi.org/ 10.1167/iovs.17-22925.

[2]. Gomes A, Fernandes E, at al. Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods JLFC (2005) 65:45–80.


H2DCFDA(DCFH-DA) is a redox-sensitive fluorescent probe, which could be used to measure intracellular reactive oxygen species levels.[1] The most popular method used to measure the level of cellular ROS formation is 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA(DCFH-DA)) assay. 

The fluorogenic dye H2DCFDA(DCFH-DA) was used to detect ROS production. Usually, after diffusion into the cell, H2DCFDA(DCFH-DA) is deacetylated by cellular esterases into a non-fluorescent compound that is subsequently oxidized by ROS into 2′,7′-dichlorofluorescein (DCF). The in vitro experiment to determine the ability of TBBPA alone to stimulate the conversion of H2DCFDA(DCFH-DA) to its fluorescent product DCF was conducted in a cell-free model. Dilution of 5 μM H2DCFDA(DCFH-DA) and increasing concentrations of TBBPA (0.1–100 μM) were added to 96-well plates containing PBS buffer without Ca2+ and Mg2+ or serum-free DMEM/F12 or DMEM/F12 supplemented with 5 % FBS in the final volume of 100 μL. The fluorescence was measured 30 and 60 min after the addition of TBBPA. The deacetylated and oxidized version of H2DCFDA(DCFH-DA): DCF ‘s fluorescence was detected at 485 and 535 nm of maximum excitation and emission spectra, respectively. This in vitro study examined the impact of TBBPA on H2DCFDA(DCFH-DA) fluorescence without cells in PBS buffer, DMEM/F12, and DMEM/F12 with 5 % of FBS media. The obtained results showed that TBBPA in all tested concentrations interacted with H2DCFDA(DCFH-DA) in PBS buffer and caused a significant increase in fluorescence. H2DCFDA(DCFH-DA) assay cannot be used in cell culture experiments with TBBPA. Results suggested that the data regarding TBBPA-stimulated ROS production in cell culture models using the H2DCFDA(DCFH-DA) assay should be revised using a different method. [3]

[1]. Park JH, Moon S-H, Kang DH, et al. Diquafosol sodium inhibits apoptosis and inflammation of corneal epithelial cells via activation of Erk1/2 and RSK: in vitro and in vivo dry eye model. Invest Ophthalmol Vis Sci. 2018;59:5108–5115. doi.org/ 10.1167/iovs.17-22925.
[2]. Szychowski KA, Rybczyńska-Tkaczyk K, et al. Tetrabromobisphenol A (TBBPA)-stimulated reactive oxygen species (ROS) production in cell-free model using the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay-limitations of method. Environ Sci Pollut Res Int. 2016 Jun;23(12):12246-52.
[3]. Gomes A, Fernandes E, at al. Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods JLFC (2005) 65:45–80

Chemical Properties

Cas No. 4091-99-0 SDF
Canonical SMILES O=C(O)C1=CC=CC=C1C2C3=C(OC4=C2C=C(Cl)C(OC(C)=O)=C4)C=C(OC(C)=O)C(Cl)=C3
Formula C24H16Cl2O7 M.Wt 487.29
Solubility ≥ 150 mg/mL in DMSO(307.82 mM); 14.29 mg/mL in Ethanol(29.33 mM); < 0.1 mg/mL in Water(insoluble) Storage Store at -20°C, protect from light
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.
Shipping Condition Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request

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Research Update

Identification of ROS using oxidized DCFDA and flow-cytometry

Cells constantly generate reactive oxygen species (ROS) during aerobic metabolism. The ROS generation plays an important protective and functional role in the immune system. The cell is armed with a powerful antioxidant defense system to combat excessive production of ROS. Oxidative stress occurs in cells when the generation of ROS overwhelms the cells' natural antioxidant defenses. ROS and the oxidative damage are thought to play an important role in many human diseases including cancer, atherosclerosis, other neurodegenerative diseases and diabetes. Thus, establishing their precise role requires the ability to measure ROS accurately and the oxidative damage that they cause. There are many methods for measuring free radical production in cells. The most straightforward techniques use cell permeable fluorescent and chemiluminescent probes. 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is one of the most widely used techniques for directly measuring the redox state of a cell. It has several advantages over other techniques developed. It is very easy to use, extremely sensitive to changes in the redox state of a cell, inexpensive and can be used to follow changes in ROS over time.

Dexamethasone induces osteoblast apoptosis through ROS-PI3K/AKT/GSK3β signaling pathway

Background: Osteoblasts play important roles in the process of osteogenesis and prevention of osteonecrosis. Dexamethasone (Dex), a type of glucocorticoids (GCs), induces apoptosis of osteoblasts and leads to the occurrence of non-traumatic osteonecrosis. This study aimed to explore the effects of phosphatidylinositol 3-kinase/Protein kinase 3 (PI3K/AKT) and glycogen synthase kinase 3β (GSK3β) on Dex-induced osteoblasts apoptosis.
Methods: Viabilities, proliferation, and apoptosis of primary osteoblasts and pre-osteoblast MC3T3-E1 cells after Dex treatment were detected using cell counting kit-8 (CCK-8) assay, 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, FITC-Annexin V/PI staining and western blotting, respectively. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to measure the intracellular reactive oxygen species (ROS) levels after Dex treatment. N-acetyl-l-cysteine (NAC) was used as ROS scavenger in this research. The expressions of PI3K/AKT and GSK3β in osteoblasts and MC3T3-E1 cells after Dex treatment were analyzed using western blotting and qRT-PCR, respectively. Then the effects of GSK3β knockdown on Dex-induced apoptosis of osteoblasts were explored. Alkaline phosphatase (ALP) activity assay was used to detect the role of Dex in regulating ALP activity.
Results: Dex remarkably inhibited proliferation and induced apoptosis of osteoblasts and MC3T3-E1 cells. Dex potentially attenuated the osteoblast differentiation. The intracellular ROS levels were significantly increased after Dex treatment. Dex suppressed the activation of PI3K/AKT pathway in osteoblasts and MC3T3-E1 cells by down-regulating the expressions of p-PI3K and p-AKT. The expressions of GSK3β in osteoblasts and MC3T3-E1 cells were obviously up-regulated after Dex treatment. Knockdown of GSK3β alleviated Dex-induced osteoblast and MC3T3-E1 cell apoptosis by decreasing the expressions of Bax, cleaved-caspase 3, cleaved-caspase 9 and increasing the expression of Bcl-2.
Conclusion: Our research verified that Dex induced osteoblasts apoptosis by ROS-PI3K/AKT/GSK3β signaling pathway.

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Matrine attenuates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via maintaining AMPK α/UCP2 pathway

Oxidative stress and cardiomyocyte apoptosis are involved in the pathogenesis of doxorubicin (DOX)-induced cardiotoxicity. Matrine is well-known for its powerful anti-oxidant and anti-apoptotic capacities. Our present study aimed to investigate the effect of matrine on DOX-induced cardiotoxicity and try to unearth the underlying mechanisms. Mice were exposed with DOX to generate DOX-induced cardiotoxicity or normal saline as control. H9C2 cells were used to verify the effect of matrine in vitro. DOX injection triggered increased generation of reactive oxygen species (ROS) and excessive cardiomyocyte apoptosis, which were significantly mitigated by matrine. Mechanistically, we found that matrine ameliorated DOX-induced uncoupling protein 2 (UCP2) downregulation, and UCP2 inhibition by genipin could blunt the protective effect of matrine on DOX-induced oxidative stress and cardiomyocyte apoptosis. Besides, 5'-AMP-activated protein kinase α2 (Ampkα2) deficiency inhibited matrine-mediated UCP2 preservation and abolished the beneficial effect of matrine in mice. Besides, we observed that matrine incubation alleviated DOX-induced H9C2 cells apoptosis and oxidative stress level via activating AMPKα/UCP2, which were blunted by either AMPKα or UCP2 inhibition with genetic or pharmacological methods. Matrine attenuated oxidative stress and cardiomyocyte apoptosis in DOX-induced cardiotoxicity via maintaining AMPKα/UCP2 pathway, and it might be a promising therapeutic agent for the treatment of DOX-induced cardiotoxicity.

Schisandrin A restrains osteoclastogenesis by inhibiting reactive oxygen species and activating Nrf2 signalling

Objectives: Intracellular reactive oxygen species (ROS) induced by receptor activator of NF-kB ligand (RANKL) has been proven to be a critical factor in the development of osteoclasts. This study aimed to prove that schisandrin A (Sch), a novel anti-oxidant compound, is able to suppress osteoclastogenesis and prevent bone loss in ovariectomized (OVX) mice by suppressing ROS via nuclear factor erythroid 2-related factor (Nrf2).
Material and methods: Micro-CT was used to detect bone formation. The effects of Sch on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced reactive oxygen species (ROS) were measured by dihydroethidium (DHE) staining in vivo and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in vitro. Immunofluorescence staining was used to detect the expression of Nrf2 in vivo. siRNA was used to evaluate the effect of Nrf2 in osteoclastogenesis.
Results: Sch suppresses RANKL-induced ROS production by regulating nuclear factor erythroid 2-related factor (Nrf2) in vitro and vivo. Mechanistically, Sch enhances the expression of Nrf2 by regulating the degradation of Nrf2. Further, Sch suppresses phosphorylation of P65 and its nuclear translocation, as well as the degradation of IκBα. Collectively, our findings reveal that Sch protects against OVX-induced bone loss by suppressing ROS via Nrf2.
Conclusions: Our results showed the potential of anti-oxidant compound schisandrin A in the treatment of osteoporosis, highlighting Nrf2 as a novel promising target in osteoclast-related disease.

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