Intracellular ROS generation was evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA) assay (Sigma, St Louis, MO, USA), which is a specific probe for hydrogen peroxide to form fluorescent dichlorofluorescein [9 (link)]. All groups were added with stimulant H2O2 (100 μM) except the vehicle group (0.5% DMSO) prior to treatment with 10 μM DCFH-DA at 37°C for 30 min. After incubation, cells were immediately submitted to fluorescence microscopy or flow cytometry and estimated using FL-1 channel.
Dcfh da assay
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DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) is a cell-permeant fluorogenic probe used to detect the presence of reactive oxygen species (ROS) within cells. It is a non-fluorescent compound that becomes fluorescent upon oxidation by ROS, allowing for the quantification of these species.
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Infinite m200 microplate reader, by Tecan (3 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Copper nanoparticles, by US Research Nanomaterials (1 mentions)
6890n gas chromatograph, by Agilent Technologies (1 mentions)
5973 mass selective detector, by Agilent Technologies (1 mentions)
Varioskan flash, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by Tecan (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
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12 protocols using dcfh da assay
1
Intracellular ROS Measurement using DCFH-DA
2
Enzymatic Biosensor Design and Characterization
3
Intracellular ROS Quantification by DCFH-DA Assay
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The level of intracellular reactive oxygen species (ROS) was measured using the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay (Sigma-Aldrich, USA), in which the intensity of fluorescence is proportional to ROS level. HK-2 cells were first seeded on 6-well plates. When they reached 70 to 80% confluence, they were treated with the relevant medium for 48 h. Then, cells were washed twice with PBS and DCFH-DA was added. Fluorescence was measured using a fluorescence microscope, after incubation for 20 min in the dark at 37°C.
4
Quantifying Intracellular ROS and Tyrosine Isomers
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2′,7′-Dichlorofluorescin diacetate (DCFH-DA) is a cell-permeable ROS-specific nonfluorescent probe. The DCFH-DA is hydrolysed intracellularly, and later it is oxidized by ROS into 2′-7′dichlorofluorescein. Intracellular steady-state ROS levels were detected using the DCFH-DA assay (Sigma-Aldrich, Istanbul, Turkey). The cells were treated PAX and RESV for 24 h. After washing the cells, they were incubated with DCFH-DA (10 μM) for 25 min at 37°C in the dark. The cells were washed again and analyzed (excitation = 485 nm, emission = 535 nm) using the microplate reader (Infinite Pro 200). The data were presented as fold increase normalized to control.
We also measured o- and m-tyrosine as previously described [65 (link)]. Briefly, phenylalanine (313 pmol and 50 nmol) and o- and m-tyrosine (313 fmol to 50 pmol) were used as calibration standards. Labeled [13C6]-phenylalanine was used as an internal standard. Each standard and sample (1 μl) was injected into the GC/MS using a pressure pulsed splitless loading 6890 N gas chromatograph interfaced to a 5973 mass selective detector (Agilent technologies), with methane as the ECNI reagent gas. o- and m-Tyrosine levels were expressed relative to phenylalanine levels.
We also measured o- and m-tyrosine as previously described [65 (link)]. Briefly, phenylalanine (313 pmol and 50 nmol) and o- and m-tyrosine (313 fmol to 50 pmol) were used as calibration standards. Labeled [13C6]-phenylalanine was used as an internal standard. Each standard and sample (1 μl) was injected into the GC/MS using a pressure pulsed splitless loading 6890 N gas chromatograph interfaced to a 5973 mass selective detector (Agilent technologies), with methane as the ECNI reagent gas. o- and m-Tyrosine levels were expressed relative to phenylalanine levels.
5
Quantifying Intracellular ROS in Breast Cancer
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The level of intracellular ROS was determined using dichlorodihydrofluorescein diacetate (DCFH-DA) assay, (Sigma, St. Louis, MO, USA). After diffusion through the cell membrane, DCFH-DA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by intracellular ROS into a fluorescent 2′,7′-dichlorofluorescein (DCF). The MDA-MB-231 and ZR-75-1 cells (1 × 104 cells per well) were seeded in 200 μL of DMEM or RPMI in 96-well black plates. After 24 h, DMEM or RPMI was removed and the cells were stained with 10 μM of DCFH-DA in PBS at 37 °C, 5% CO2, for 45 min. Then, the dye was removed and replaced with the rGO suspensions in DMEM or RPMI, at 50 μg/mL or 100 μg/mL concentrations and incubated for 24 h and 48 h. The DCF fluorescence intensity was measured by Infinite M200 microplate reader (Tecan, Männedorf, Switzerland), at the excitation wavelength of 485 nm and the emission wavelength of 535 nm. The intracellular ROS generation in rGO-stimulated breast cancer cells was shown as the intensity of fluorescence of the DCF.
6
Intracellular ROS Detection in Breast Cancer
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The level of intracellular ROS was determined using the dichlorodihydrofluorescein diacetate (DCFH-DA) assay (Sigma, St. Louis, MO, USA). After diffusion through the cell membrane, DCFH-DA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by intracellular ROS into fluorescent 2′,7′-dichlorofluorescein (DCF). MDA-MB-231 and ZR-75-1 cells (1 × 104 cells per well) were seeded in 200 μL of DMEM or RPMI in 96-well black plates. After 24 h, DMEM or RPMI was removed, and the cells were stained with 10 μM of DCFH-DA in PBS at 37°C, 5% CO2, for 45 min. Then, the dye was removed and replaced with the rGO suspensions in DMEM or RPMI at 100 μg/mL, MG-132 (5 µM) or a mix consisting of rGO (100 μg/mL) and MG-132 (5 µM) and incubated for 48 h. The DCF fluorescence intensity was measured by Infinite M200 microplate reader (Tecan, Männedorf, Switzerland) at the excitation wavelength of 485 nm and the emission wavelength of 535 nm. The intracellular ROS generation in breast cancer cells stimulated by rGO, MG-132 (5 µM) or a mix consisting of rGO (100 μg/mL) and MG-132 (5 µM) was shown as the intensity of fluorescence of the DCF.
7
Intracellular ROS Detection in LBC3 Cells
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The level of intracellular ROS was determined using dichlorodihydrofluorescein diacetate (DCFH-DA) assay, (Sigma, St. Louis, MO, USA). After diffusion through the cell membrane, DCFH-DA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by intracellular ROS into a fluorescent 2′,7′-dichlorofluorescein (DCF). The LBC3 cells (10 × 105) were seeded in 200 μL of DMEM in 96-well black plates. After 24 h, DMEM was removed and the cells were stained with 10 μM of DCFH-DA in PBS at 37 °C, 5% CO2 incubator, for 45 min. Then, the dye was removed and replaced with the 5–15 nm SiNPs suspensions in DMEM, at 50 or 100 μg/mL concentrations and incubated for 24 and 48 h. The DCF fluorescence intensity was measured by Infinite M200 microplate reader (Tecan, Salzburg, Austria), at the excitation wavelength of 485 nm and the emission wavelength of 535 nm. The intracellular ROS generation in SiNPs-stimulated LBC3 cells was shown as the intensity of fluorescence of the DCF.
8
Intracellular ROS Measurement by DCFH-DA Assay
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Intracellular ROS was determined by fluorometric assay (DCFH-DA assay, Sigma, USA). Cells were plated in 6-well plates (1 × 106 per well) and incubated with glucose (33.3 mM) and/or MK801 (50 μM) for 72 h. Cells were loaded with 10 μM DCFH-DA for 20 min at 37 °C. The generation of ROS was determined using Varioskan Flash (Thermo Fisher Scientific, USA) with excitation and emission wavelengths at 488 and 525 nm, respectively. Relative ROS content was standardized to total protein content of the cells.
9
Intracellular ROS Production Assay
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The production of intracellular reactive oxygen species (ROS) was determined using DCFH-DA assay (Sigma-Aldrich, Munich, Germany). Per well, 1 × 104 HUVECs were seeded into the 96-well black plate and incubated overnight at 37 °C until 80% confluence. After irradiation, the cells were incubated with 10 μM DCFH-DA in a serum-free medium for 30 min in the dark. Afterward, the plates were rinsed with PBS thrice to remove the free DCFH-DA. The fluorescence intensities were detected at an excitation wavelength of 488 nm and an emission wavelength of 525 nm using a microplate reader (Tecan, Männedorf, Switzerland). Results are shown as a percentage as compared to non-irradiated cells.
10
Intracellular ROS Quantification Using DCFH-DA
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The level of intracellular ROS was determined using a dichlorodihydrofluorescein diacetate (DCFH-DA) assay, (Sigma, St. Louis, MO, USA) as described in our previous studies [7 (link)].
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