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  • H2DCFDA (DCFH-DA)
H2DCFDA (DCFH-DA)的可视化放大

H2DCFDA (DCFH-DA)

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。

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  • 货号: ajce44254
  • CAS: 4091-99-0
  • 别名: 2',7'-二氯荧光素二乙酸酯,DCFH-DA; 2',7'-Dichlorodihydrofluorescein diacetate
  • 分子式: C24H16Cl2O7
  • 分子量: 487.29
  • 纯度: >98%
  • 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: Store at -20°C, protect from light
  • 库存: 现货

Background

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。

参考文献:
[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

Protocol

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.

参考文献:

[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.

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