H2DCFDA (DCFH-DA)
(Synonyms: 2',7'-二氯荧光素二乙酸酯,DCFH-DA; 2',7'-Dichlorodihydrofluorescein diacetate) 目录号 : GC30006
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。
Cas No.:4091-99-0
Sample solution is provided at 25 µL, 10mM.
- Biomolecules 13.1 (2023): 102.PMID:36671487
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Quality Control & SDS
- View current batch:
- Purity: >97.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
本方案仅提供一个指导，请根据您的具体需要进行修改。
1.制备染色液
(1)配置储存液:使用DMSO溶解H2DCFDA (DCFH-DA)，配置浓度为1-10mM的储存液。
注意:未使用的储存液分装后在-20℃或-80℃避光保存，避免反复冻融。
(2)配置工作液:用合适的缓冲液(如:无血清培养基或PBS)稀释储存液，配制浓度为1-10μM的工作液。
注意:请根据实际情况调整工作液浓度，现用现配。
2.细胞悬浮染色
(1)悬浮细胞：经4℃、1000g离心3-5分钟，弃去上清液，用PBS清洗两次，每次5分钟。
(2)贴壁细胞：使用PBS清洗两次，加入胰酶消化细胞，消化完成后经1000g离心3-5min。
(3)加入H2DCFDA (DCFH-DA)工作溶液重悬细胞，37℃避光孵育5-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
(4)孵育结束后，经1000g离心5分钟，去除上清液，加入PBS清洗2-3次，每次5分钟。
(5)用预温的无血清细胞培养基或PBS孵育细胞。
3.细胞贴壁染色
(1)在无菌盖玻片上培养贴壁细胞。
(2)从培养基中移走盖玻片，吸出过量的培养基，将盖玻片放在潮湿的环境中。
(3)从盖玻片的一角加入100μL的染料工作液，轻轻晃动使染料均匀覆盖所有细胞。
(4) 37℃避光孵育5-30分钟。不同细胞最佳孵育时间不同，请根据具体实验需求自行摸索。
(5)孵育结束后吸弃染料工作液，使用预温的培养液清洗盖玻片2~3次；
(6) 用预温的无血清细胞培养基或PBS孵育细胞。
4.显微镜检测：H2DCFDA (DCFH-DA)的最大激发/发射波长为488/525nm。
注意事项：
① 对于二乙酸酯衍生物，需要短暂复原时间让细胞内酯酶将其水解，从而让染料对氧化应激产生反应。最佳的复原时间范围很广，由于某些细胞类型通常显示极其低水平的酯酶活性；
② 将细胞暴露于实验刺激物之前，需要先测定细胞的背景荧光强度；
③ 对于流式分析，染料处理前后细胞的前向角散射和侧向角散射是不变，细胞尺寸的变化可能是加药处理或有毒反应引起的；
④ 建议对不含细胞的实验体系进行荧光检测，在不含细胞外酯酶和其他氧化酶的情况下，随着时间荧光的逐渐增加可能与瞬间水解、空气氧化以及光诱导氧化等因素有关；
⑤ 对于对照组（未加药）细胞，细胞内酶或天然抗氧化剂会清除氧自由基，经过染料加载复原时间后，健康细胞应该表现出低水平的荧光信号，且在整个实验期间相对稳定；
⑥ 可能会观察到逐渐增加(由于自氧化)或降低(由于细胞内染料丧失或光淬灭)的荧光信号；
⑦ 在不含任何刺激物或诱导剂的体系内，健康和未处理细胞突然发现强荧光，表明细胞发生死亡或一些其他的氧化事件；
⑧ 荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光淬灭；
为了您的安全和健康，请穿实验服并戴一次性手套操作。
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]
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
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。
| 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 |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.0522 mL | 10.2608 mL | 20.5217 mL |
| 5 mM | 0.4104 mL | 2.0522 mL | 4.1043 mL |
| 10 mM | 0.2052 mL | 1.0261 mL | 2.0522 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
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.