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H2DCFDA (DCFH-DA) Sale

(Synonyms: 2',7'-二氯荧光素二乙酸酯,DCFH-DA; 2',7'-Dichlorodihydrofluorescein diacetate) 目录号 : GC30006

H2DCFDA(又称DCFH-DA)是一种能够穿透细胞膜的探针,用于检测细胞内活性氧自由基(ROS),其激发波长为488纳米,发射波长为525纳米。

H2DCFDA (DCFH-DA) Chemical Structure

Cas No.:4091-99-0

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10mM (in 1mL DMSO)
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50mg
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实验参考方法

Cell experiment [1, 2]:

Cell lines

hCECs

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.

Applications

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.

References:

[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.

产品描述

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]

H2DCFDA(DCFH-DA)是一种氧化还原敏感的荧光探针,可用于测量细胞内活性氧水平。目前最流行的方法是使用2′,7′-二氯二羟荧光素酯(H2DCFDA(DCFH-DA))试剂来测量细胞ROS生成水平。

本实验使用荧光染料H2DCFDA(DCFH-DA)来检测ROS的产生。通常情况下,H2DCFDA(DCFH-DA)扩散进入细胞后,被细胞内酯酶脱乙酰化成为一种非荧光化合物,然后被ROS氧化成为2'、7'-二氯荧光素(DCF)。在无细胞模型中进行了TBBPA单独刺激将H2DCFDA(DCFH-DA)转化为其荧光产物DCF的体外实验。将5μM H2DCFDA(DCFH- DA)稀释和不断增加的TBBPA浓度(0.1–100 μM)添加到96孔板中,其中含有PBS缓冲液而没有Ca 2+和Mg 2+,或者是无血清DMEM/F12或DMEM/F12培养基,最终体积为100μL并且含有5% FBS。在加入TBBPA之后30分钟和60分钟分别测量了荧光值。去乙酰化和氧化版本的 H 2 DCFD A(DCF H - DA): DCF 的荧光在最大激发波长485nm 和发射波长535nm处检测到。这项体外研究考察了TBBPA对PBS缓冲液、DMEM/F12和含有5% FBS培养基中H2DCFDA(DCFH-DA)荧光的影响。结果表明,TBBPA在所有测试浓度下都与PBS缓冲液中的H2DCFDA(DCFH- DA)相互作用,并导致荧光显著增加。因此,在使用 H 2 DCFD A(DCF H - DA)检测法进行细胞培养实验时不能使用该方法。研究结果建议应采用不同的方法来修正关于TBBPA刺激ROS产生的细胞培养模型中使用 H 2 DCFD A(DCF H - DA)检测法所得到的数据。

References:
[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
别名 2',7'-二氯荧光素二乙酸酯,DCFH-DA; 2',7'-Dichlorodihydrofluorescein diacetate
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
分子式 C24H16Cl2O7 分子量 487.29
溶解度 ≥ 150 mg/mL in DMSO(307.82 mM); 14.29 mg/mL in Ethanol(29.33 mM); < 0.1 mg/mL in Water(insoluble) 储存条件 Store at -20°C, protect from light
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Research Update

Identification of ROS using oxidized DCFDA and flow-cytometry

Methods Mol Biol 2010;594:57-72.20072909 10.1007/978-1-60761-411-1_4

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.

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

J Vis Exp 2020 Jun 23;(160):10.3791/60682.32658187 PMC7712457

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.

Alborixin clears amyloid-β by inducing autophagy through PTEN-mediated inhibition of the AKT pathway

Autophagy 2019 Oct;15(10):1810-1828.30894052 PMC6735498

Imbalance in production and clearance of amyloid beta (Aβ) is the primary reason for its deposition in Alzheimer disease. Macroautophagy/autophagy is one of the important mechanisms for clearance of both intracellular and extracellular Aβ. Here, through screening, we identified alborixin, an ionophore, as a potent inducer of autophagy. We found that autophagy induced by alborixin substantially cleared Aβ in microglia and primary neuronal cells. Induction of autophagy was accompanied by up regulation of autophagy proteins BECN1/Beclin 1, ATG5, ATG7 and increased lysosomal activities. Autophagy induced by alborixin was associated with inhibition of the phosphoinositide 3-kinase (PI3K)-AKT pathway. A knock down of PTEN and consistent, constitutive activation of AKT inhibited alborixin-induced autophagy and consequent clearance of Aβ. Furthermore, clearance of Aβ by alborixin led to significant reduction of Aβ-mediated cytotoxicity in primary neurons and differentiated N2a cells. Thus, our findings put forward alborixin as a potential anti-Alzheimer therapeutic lead. Abbreviations: Aβ: amyloid beta; ALB: alborixin; ATG: autophagy-related; BECN1: beclin 1; DAPI: 4, 6-diamidino-2-phenylindole; DCFH-DA: 2,7-dichlorodihydrofluorescein diacetate; fAβ: fibrillary form of amyloid beta; GFAP: glial fibrillary acidic protein; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MTOR: mechanistic target of rapamycin kinase; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SQSTM1: sequestosome 1; TMRE: tetramethylrhodamine, ethyl ester.

Active oxygen chemistry within the liposomal bilayer. Part IV: Locating 2',7'-dichlorofluorescein (DCF), 2',7'-dichlorodihydrofluorescein (DCFH) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in the lipid bilayer

Chem Phys Lipids 2004 Aug;131(1):123-33.15210370 10.1016/j.chemphyslip.2004.04.006

2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is commonly used to detect the generation of reactive oxygen intermediates and for assessing the overall oxidative stress in toxicological phenomenon. It has been suggested that DCFH-DA crosses the cell membrane, subsequently undergoing deacetylation by intracellular esterases. The resulting 2',7'-dichlorodihydrofluorescein (DCFH) is proposed to react with intracellular hydrogen peroxide or other oxidizing ROS to give the fluorescent 2',7'-dichlorofluorescein (DCF). Using an NMR chemical shift-polarity correlation, we have determined that DCFH-DA and DCFH are located well within the lipid bilayer and certainly not at the interface. These results, therefore, put into serious question the proposed ability of DCFH to come in contact with the aqueous phase and thereby interact with aqueous intracellular ROS and components. However, H2O2 and superoxide can cross or at least penetrate the lipid bilayer and react with certain lipophilic substrates. This may well describe the mode of reaction of these and other ROS with DCFH.

Gut microbiota modulates osteoclast glutathione synthesis and mitochondrial biogenesis in mice subjected to ovariectomy

Cell Prolif 2022 Mar;55(3):e13194.35080066 PMC8891549

Objectives: Osteoporosis is a common bone disease in the elderly mainly regulated by osteoblasts (OBs) and osteoclasts (OCs). The gut microbiota has been recognized as an important factor in many physiological and pathological processes in the host. Thus, we hypothesize that the gut microbiota is necessary for postmenopausal osteoporosis and that germ-free (GF) mice are protected from osteoporosis. Material and methods: Osteoporosis models were established by performing ovariectomy (OVX) in mice. Bone mass was measured by micro-CT, and gut microbiota were assessed by 16s rDNA sequencing. Reactive oxygen species (ROS) were detected by dihydroethidium (DHE) staining in vivo and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in vitro. Results: Firmicutes and Bacteroidetes in the intestine are pivotal in OC differentiation, and the Firmicutes/Bacteroidetes ratio (F/B ratio) is a specific indicator of osteoporosis. Furthermore, we found that Firmicutes and Bacteroidetes affect the de novo synthesis of glutathione (GSH) by regulating its key enzyme glutamate-cysteine ligase catalytic subunit (Gclc) and inhibiting mitochondrial biogenesis and ROS accumulation via the cAMP response element-binding (CREB) pathway. In addition, supplementing OVX mice with the probiotic Lactobacillus salivarius LI01 from the Firmicutes phylum prevented osteoporosis. Conclusions: Our results reveal that GSH plays a vital role in OVX-induced bone loss, and probiotics that affect GSH metabolism are potential therapeutic targets for overcoming osteoporosis.