Intracellular ROS were quantified using the fluorogenic probe, 2′,7′–dichlorodihydrofluorescein diacetate (DCF-DA) assay (Sigma-Aldrich), according to the manufacturer’s protocol. Cells were seeded in a 12-well plate at a density of 1 × 105 cells/well and incubated for 24 h. After different treatments (see Section 2.2 for details), the well plates were replaced with 100 mM DCF-DA diluted in DMEM for 30 min. The cells were washed with fresh DMEM and incubated for an additional 10 min to allow for excitation of DCF fluorescence. The resulting fluorescence was photographed and recorded with a fluorescence analyzer (JuLI-FL, Pleasanton, CA, USA) at an excitation/emission spectra of 504/524 nm. The intensities were analyzed using ImageJ software (ImageJ v1. 46a; NIH, Bethesda, MD, USA) and calculated as the fold change in the CTRL group.
Dcf da assay
Manufactured by Merck Group
The DCF-DA (2',7'-Dichlorofluorescin diacetate) assay is a widely used fluorometric method for detecting and measuring the presence of reactive oxygen species (ROS) within cells. The assay is based on the principle that DCF-DA, a non-fluorescent compound, is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF) upon reaction with ROS. This change in fluorescence intensity can be quantified and used as an indicator of the cellular oxidative status.
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3 protocols using dcf da assay
1
Intracellular ROS Quantification using DCF-DA Assay
2
ROS Detection in Cells Using DCFDA Assay
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Reactive oxygen species (ROS) detection was undertaken using the fluorescent 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFDA) assay (Sigma-Aldrich). Cells were seeded at 1 × 105 and incubated for 24 h. On the day of treatment, the test chemical and positive control were diluted accordingly. The 4000× DCFDA stock was diluted to 1× DCFDA, and 100 µl was added to each well in a 96-well plate. The cell flasks were treated with CdCl2, and a 100 µl aliquot was immediately added to the 96-well plate. Fluorescence readings were taken using a FLUOstar Omega Multimode microplate reader (BMG LABTECH Ltd, UK) with excitation/emission set at 485/535 at various time points from the time of dosing; 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h. Hydrogen peroxide (50 μM) was used as a positive control in all experiments.
3
ROS Quantification via DCF-DA Assay
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The level of reactive oxygen species (ROS) formation in cells was determined by a 2′,7′-dichlorofluorescin diacetate
(DCF-DA) assay (Sigma Aldrich). DCF-DA is a cell-permeable non-fluorescent probe, which is de-esterified intracellularly and turns into highly fluorescent 2′,7′-dichlorofluorescein upon oxidation. Cells were seeded on a 96-well black plate with a clear bottom (Eppendorf); after overnight incubation, the medium was replaced with medium containing nanomaterials at all tested concentrations. The ROS level was measured after 2 h of cells' incubation with nanomaterials, when the medium was removed and 100 µL of 10 µM DCF-DA in PBS was added to each well. After 45 min, the solution was removed, and the fluorescence intensity was read at λ = 520 nm after excitation at λ = 480 nm, using a microplate reader (Tecan Group Ltd.). The level of ROS was expressed as the percentage of ROS level in the control group, which was designated as 100%. Calculations were performed with the following formula:
where MFI test is the mean fluorescence intensity of wells exposed to the nanomaterials, and MFI control is the mean absorbance of control wells.
(DCF-DA) assay (Sigma Aldrich). DCF-DA is a cell-permeable non-fluorescent probe, which is de-esterified intracellularly and turns into highly fluorescent 2′,7′-dichlorofluorescein upon oxidation. Cells were seeded on a 96-well black plate with a clear bottom (Eppendorf); after overnight incubation, the medium was replaced with medium containing nanomaterials at all tested concentrations. The ROS level was measured after 2 h of cells' incubation with nanomaterials, when the medium was removed and 100 µL of 10 µM DCF-DA in PBS was added to each well. After 45 min, the solution was removed, and the fluorescence intensity was read at λ = 520 nm after excitation at λ = 480 nm, using a microplate reader (Tecan Group Ltd.). The level of ROS was expressed as the percentage of ROS level in the control group, which was designated as 100%. Calculations were performed with the following formula:
where MFI test is the mean fluorescence intensity of wells exposed to the nanomaterials, and MFI control is the mean absorbance of control wells.
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