2',7'-Dichlorofluorescein diacetate
(Synonyms: 双氯荧光黄乙酸乙酯,DCFH2-DA) 目录号 : GC42079
A cell-permeable fluorogenic probe to quantify ROS and NO
Cas No.:2044-85-1
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >98.00%
- 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2',7'-Dichlorofluorescein diacetate（DCFH2-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°C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1-100μM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°C、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DCFH2-DA工作溶液重悬细胞，室温避光孵育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重悬细胞，通过荧光显微镜或流式细胞术观察。
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) 室温避光孵育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。
4.显微镜检测：DCFH2-DA的最大吸收/发射波长为492/515nm。
注意事项：
1）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。
2）为了您的安全和健康，请穿实验服并戴一次性手套操作。
2',7'-Dichlorofluorescein diacetate is as a cell-permeable fluorogenic probe to quantify reactive oxygen species (ROS) and nitric oxide (NO). It is rapidly de-esterified in cells is oxidized to form fluorescent 2',7'-dichlorofluorescein. 2'7-Dichlorofluorescein displays excitation/emission spectra of 492/515 nm.
| Cas No. | 2044-85-1 | SDF | |
| 别名 | 双氯荧光黄乙酸乙酯,DCFH2-DA | ||
| Canonical SMILES | CC(OC1=C(Cl)C=C(C2(C(C=CC=C3)=C3C(O2)=O)C(C=C(Cl)C(OC(C)=O)=C4)=C4O5)C5=C1)=O | ||
| 分子式 | C24H14Cl2O7 | 分子量 | 485.3 |
| 溶解度 | 0.1 M Na2CO3: 5 mg/ml,DMF: 25 mg/ml,DMSO: 33 mg/ml,Ethanol: 25 mg/ml,PBS (pH 7.2): 20 µ g/ml | 储存条件 | Store at 2-8°C |
| 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 | 2.0606 mL | 10.3029 mL | 20.6058 mL |
| 5 mM | 0.4121 mL | 2.0606 mL | 4.1212 mL |
| 10 mM | 0.2061 mL | 1.0303 mL | 2.0606 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。
Optimal Use of 2',7'-Dichlorofluorescein diacetate in Cultured Hepatocytes
Methods Mol Biol 2022;2451:721-747.PMID:35505044DOI:10.1007/978-1-0716-2099-1_39.
Oxidative stress is a state that arises when the production of reactive transients overwhelms the cell's capacity to neutralize the oxidants and radicals. This state often coincides with the pathogenesis and perpetuation of numerous chronic diseases. On the other hand, medical interventions such as radiation therapy and photodynamic therapy generate radicals to selectively damage and kill diseased tissue. As a result, the qualification and quantification of oxidative stress are of great interest to those studying disease mechanisms as well as therapeutic interventions. 2',7'-Dichlorodihydrofluorescein-diacetate (DCFH2-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH2-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2',7'-dichlorodihydrofluorescein (DCFH2). The nonfluorescent DCFH2 is subsequently oxidized by reactive transients to form the fluorescent 2',7'-dichlorofluorescein (DCF). The use of DCFH2-DA in hepatocyte-derived cell lines is more challenging because of membrane transport proteins that interfere with probe uptake and retention, among several other reasons. Cancer cells share some of the physiological and biochemical features with hepatocytes, so probe-related technical issues are applicable to cultured malignant cells as well. This study therefore analyzed the in vitro properties of DCFH2-DA in cultured human hepatocytes (HepG2 cells and differentiated and undifferentiated HepaRG cells) to identify methodological and technical features that could impair proper data analysis and interpretation. The main issues that were found and should therefore be accounted for in experimental design include the following: (1) both DCFH2-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH2 oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH2-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH2-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH2-DA was applied to visualize and quantify oxidative stress in real time in HepG2 cells subjected to anoxia/reoxygenation as a source of reactive transients.
Quantification of Reactive Oxygen Species Using 2',7'-Dichlorofluorescein diacetate Probe and Flow-Cytometry in Müller Glial Cells
J Vis Exp 2022 May 13;(183).PMID:35635447DOI:10.3791/63337.
The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, β-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since Müller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.
Measurement of Intracellular ROS in Caenorhabditis elegans Using 2',7'-Dichlorodihydrofluorescein Diacetate
Bio Protoc 2018 Mar 20;8(6):e2774.PMID:29744374DOI:10.21769/BioProtoc.2774.
Reactive oxygen species (ROS) are generated during normal metabolic processes under aerobic conditions. Since ROS production initiates harmful radical chain reactions on cellular macromolecules, including lipid peroxidation, DNA mutation, and protein denaturation, it has been implicated in a wide spectrum of diseases such as cancer, cardiovascular disease, ischemia-reperfusion and aging. Over the past several decades, antioxidants have received explosive attention regarding their protective potential against these deleterious reactions. Accordingly, many analytical methodologies have been developed for the evaluation of the antioxidant capacity of compounds or complex biological samples. Herein, we introduce a simple and convenient method to detect in vivo intracellular ROS levels photometrically in Caenorhabditis elegans using 2',7'-Dichlorofluorescein diacetate (H2DCFDA), a cell permeant tracer.
Are synapses targets of nanoparticles?
Biochem Soc Trans 2010 Apr;38(2):536-8.PMID:20298217DOI:10.1042/BST0380536.
The last few years have been marked by real breakthroughs in the field of nanotechnology. Application of nanoparticles was proposed for diagnosis and treatment of different central nervous system diseases. Exposure to nanoparticles in vivo increases the risk of onset of neurodegenerative diseases and nanoparticles are apparently able to kill neurons in vitro. We suggested that presynaptic terminals of neurons are another target for nanoparticles, beyond the already established microglial cells. Ferritin was chosen as a prototypic nanoparticle model. We found that even a high concentration of ferritin, 800 microg/ml, was not able to induce spontaneous release of [(14)C]glutamate. In contrast, [(14)C]glutamate uptake was inhibited by ferritin in a dose-dependent fashion. As a next step, the influence of ferritin on the formation of reactive oxygen species was monitored using the fluorescent dye DCFH-DA (2',7'-Dichlorofluorescein diacetate). It was shown that ferritin leads to a dose-dependent formation of free radicals. We found that the ferritin-mediated changes in glutamatergic neurotransmission at presynaptic endings can result in neuronal damage and finally neurodegeneration.
A microplate-based DCFH-DA assay for the evaluation of oxidative stress in whole semen
Heliyon 2022 Sep 15;8(9):e10642.PMID:36158085DOI:10.1016/j.heliyon.2022.e10642.
Aims: The well-documented relationship between sperm oxidation and male infertility strongly encourages the development of assays for reactive oxygen species detection in semen samples. The present study aims to apply the microplate-based 2',7'-Dichlorofluorescein diacetate assay to the evaluation of oxidative stress in unprocessed whole semen, thus avoiding sample centrifugations and other manipulations that may cause significant reactive oxygen species increments. Main methods: The fluorescence assay consisted in the quantification of both intracellular and extracellular reactive oxygen species levels in unwashed semen specimens by using the probe 2',7'-Dichlorofluorescein diacetate into a 96-well plate. The method was useful for the preliminary assessment of the oxidation levels of whole semen samples from men undergoing standard sperm analysis as well as to evaluate the effect of some pro-glutathione molecules on semen oxidative status. Key findings: The 2',7'-Dichlorofluorescein diacetate assay was successfully adapted to the evaluation of oxidative stress in whole semen, effectively revealing the perturbation of the redox homeostasis of the sample. Accordingly, specimens with abnormal sperm parameters (n = 10) presented oxidation indexes significantly higher than those with normospermia (n = 10) [7729 (range 3407-12769) vs. 1356 (range 470-2711), p < 0.001]; in addition, semen oxidation indexes negatively correlated to sperm motility and morphology. Noteworthy, whole semen exposure to pro-glutathione compounds led to reduced semen oxidation levels and sperm protection against oxidative damage. Significance: Based on our pilot experimental data, the microplate-based 2',7'-Dichlorofluorescein diacetate assay appears to be a convenient method for the detection of reactive oxygen species levels in whole semen samples, avoiding artifacts due to semen centrifugation steps. At the same time, the test could be a helpful tool for the basic and quick screening of antioxidant molecules able to preserve semen quality.