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.

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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. So far, it has been shown that TBBPA can increase ROS production in different cell culture models and, in that way, cause apoptosis.^[2]

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