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。