To assess ROS production under TBI conditions, fungal hyphae incubated for 24 h and then treated with or without AgNPs for another 48 h were stained with 10 μM DCFH-DA (S0033S, Beyotime, Shanghai, China). Hyphae were stained with DCFH-DA dye at room temperature for 30 min and then observed in a bight/fluorescence field of view at the excitation and emission wavelengths of 488 and 525 nm, respectively, under a Zeiss LSM780 confocal microscope (Gottingen). Each experiment was conducted in triplicate.
Dcfh da
Manufactured by Zeiss
Sourced in Germany
DCFH-DA is a fluorescent probe used to detect the presence of reactive oxygen species (ROS) in biological samples. It is a cell-permeable dye that is converted to a fluorescent compound upon oxidation, allowing for the measurement of oxidative stress levels in cells.
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Lab products found in correlation
Dcfh da, by Merck Group (4 mentions)
Dcfh da, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Dcfh da, by Beyotime (2 mentions)
Lsm 710 laser confocal microscope, by Zeiss (2 mentions)
Mitosox red, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Daf fm, by Merck Group (1 mentions)
Mitosox red, by Zeiss (1 mentions)
Facsarray bioanalyzer, by BD (1 mentions)
Stereo lumar v12, by Zeiss (1 mentions)
Axiovert inverted microscope, by Zeiss (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Dcfh da, by Abcam (1 mentions)
Mitotracker red, by Zeiss (1 mentions)
Confocal laser scanning microscope, by Zeiss (1 mentions)
Mitotracker red, by Beyotime (1 mentions)
Dcfh da fluorescent probe, by Beyotime (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
11 protocols using dcfh da
1
Evaluating Oxidative Stress in Fungal Hyphae
2
Measuring ROS in Embryonic Stem Cells
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After the indicated treatment, the production of ROS was measured by staining ESCs with the fluorescent dye DCFH-DA (Thermo Fisher Scientific, USA). Briefly, the cells were washed 3 times and loaded with 5 μM DCFH-DA, then incubated for 30 min at a temperature of 37°C in darkness. A confocal microscope (Zeiss, Germany) was used to quantify the resulting DCFH-DA fluorescence under 488 nm excitation light.
3
Quantifying Cellular Reactive Oxygen Species
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Cellular ROS was detected using specific ROS probes by confocal microscope and flow cytometry. For flow cytometric measurement, cells were incubated with 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM, Sigma, D6883) or dihydroethidium (DHE, 5 μM, Sigma, D7008) for 30 min at 37 °C, after which the cells were washed and suspended in ice-cold PBS and analyzed for fluorescence intensity using a 485 nm excitation beam (FACS ArrayBioanalyzer, BD biosciences). Flow Jo 7.6 was utilized to quantify the mean fluorescence intensity. For confocal measurement, ROS measurement was assayed by DCFH-DA, DHE, MitoSOX TM Red mitochondrial superoxide indicator (MitoSOX Red, Invitrogen, M36008), or 4-amino-5-methylamino-29,79-difluorofluorescein (DAF-FM, Sigma, D2321), according to manufacturer’s instruction. Briefly, cells were loaded with DCFH-DA (10 μM for 30 min), DHE (5 μM for 30 min), MitoSOX Red (2.5 μM for 10 min), or DAF-FM (10 μM for 30 min), washed with ice cold HBSS (Hank’s Balanced Salt Solution, pH 7.2), and then observed under a Zeiss LSM710 laser confocal microscope (Carl Zeiss, Germany) equipped with Zen software to process the images.
4
Visualizing H2O2 Accumulation in Lemna trisulca
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Accumulation of H2O2 in L. trisulca was detected by staining with dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich), as described by Małecka et al. [102 ]. Fresh plants were submerged in 4 μM DCFH-DA dissolved in dimethylsulfoxide (DMSO) in 50 mM potassium phosphate buffer, pH 7.4, for 2 h. The fronds were washed twice with the loading buffer and then observed under a fluorescence stereomicroscope SteREO Lumar V12 (Zeiss, Jena, Germany) with a filter set Lumar 38 HE (excitation 470/40 nm, emission 525/50 nm) and objective NeoLumar 0.8×. Images were analyzed with AxioVision software, version 4.2 (Zeiss, Jena, Germany). Microscopic analyses were based on 3 replications of 10 plants each.
5
Reactive Oxygen Species Quantification
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Reactive oxygen species production was evaluated as previously described (Yan et al., 2009 (link)). In brief, yeast cells were incubated with 20 mg/L of 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Sigma–Aldrich) for 30 min at 35°C, at 150 rpm. Cells were washed once (2,655 × g for 5 min at room temperature; 5417R, Eppendorf) and resuspended in phosphate-buffered saline (PBS, Sigma–Aldrich); afterward, cells were treated with the antimicrobials as described in “Functional characterization of drug interactions” section. FI was determined at FL1 (530 nm). ROS production was calculated by subtracting the FI value displayed by cells treated with antimicrobials from that of cells treated with both antimicrobials and DCFH-DA.
Simultaneously, 10 μL of the treated cell suspension were placed in a microscope slide for further analysis under fluorescence microscopy, in a Carl Zeiss Axiovert inverted microscope, using laser wavelength of 488 nm.
Simultaneously, 10 μL of the treated cell suspension were placed in a microscope slide for further analysis under fluorescence microscopy, in a Carl Zeiss Axiovert inverted microscope, using laser wavelength of 488 nm.
6
Detecting Intracellular ROS in Leptospire-Infected Cells
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The intracellular ROS levels in leptospire-infected cells were detected by Dichlorofluorescein diacetate (DCFH-DA) (Sigma), a ROS specific fluorescent dye (Hu et al., 2013 (link)). Briefly, the leptospire-infected cells were incubated in medium containing 5 mM DCFH-DA for 30 min at 37°C, and then the fluorescence intensity reflecting ROS levels was detected by laser confocal microscopy (LSM510-Meta, Zeiss, Germany).
7
Quantifying ROS Generation Using DCFH-DA
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The generation of ROS was determined using 2′-7′dichlorofluorescin diacetate (DCFH-DA, Beyotime Biotechnology Co., Shanghai, China), which can be oxidized to dichlorofluorescein (DCF) with high fluorescence in cells. Cells were starved for 6 h in serum-free DMEM medium and then pretreated with or without Hyp (2 or 5 mmol/L) for 12 h at 37 °C before being stimulated with 4-HNE (40 μmol/L) for 3 h in the presence or absence of Hyp. Subsequently, the cells were exposed to 10 μmol/L DCFH-DA for an additional 30 min at 37 °C and observed under a fluorescence microscope (Zeiss, Germany). DCF fluorescence intensity was quantified using Image J software (NIH, USA).
8
Metformin Modulates Cellular Redox and Mitochondrial Membrane Potential
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The ROS levels were determined using 2, 7-dichlorodihydrofluorescein diacetate (DCFH-DA, Abcam), a fluorogenic dye that measures intracellular hydroxyl and peroxyl activities. The fluorescent dye Mito-Tracker Red (#C1071S, Beyotime, Shanghai, China) was used to monitor the changes in MMP. Briefly, PGL626 cells (5 × 105 cells/well) were seeded in 24-well plates to achieve about 80% confluence. Subsequently, the medium was replaced with fresh medium with different metformin concentrations of 0, 10 and 20 mM for 48 h. At the end of incubation, the cells were incubated with 10 mM of DCFH-DA or 1 μM Mito-Tracker Red in serum-free medium for 20–30 min in the dark at room temperature. The residual reagent was removed by washing with PBS three times. The fluorescence of DCFH-DA (488 nm excitation/525 nm emission) as well as the Mito-Tracker Red (530 nm excitation/590 nm emission) were captured by a Zeiss confocal laser scanning microscope.
9
Quantifying Nitric Oxide and ROS
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NO of cell supernatant was detected using Griess Reagent (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions. Samples and standards were added into a 96-well plate, and then Griess Reagent I and II were added successively. The absorbance was determined at a wavelength of 540 nm using a microplate reader. ROS was determined by a Reactive Oxygen Species Assay Kit (Beyotime Institute of Biotechnology). In brief, after washing, cells were incubated with fluorescent probe DCFH-DA (10 μM, Beyotime Institute of Biotechnology) for 20 min at 37 °C in an incubator. Then, the extracellular DCFH-DA was cleared, and images were obtained using a fluorescence microscope (Carl Zeiss) at the 488 nm excitation wavelength and 525 nm emission wavelength.
10
Quantifying LDL Receptor Abundance and ROS in CRC
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Anti-LDL Receptor antibody (ab30532) (Abcam, Cambridge, MA, USA) (1:100 dilution) was used to evaluate the abundance of LDL Receptor protein in the 90 CRC samples. Briefly, the extent of immunostaining was defined as 0 for 0% positive cells under the microscope, 1 for 1 -25% positive cells, 2 for 26 -50% positive cells, 3 for 51 -75% positive cells and 4 for ≥ 76% positive cells. Intensity was scored as 0 for absence of staining, 1 for weak, 2 for moderate, and 3 for strong staining.
ROS assays SW480, LoVo and RKO cell lines were treated with LDL (100 μg/ml) for 36 h, followed by ROS measurement using dichloro-dihydro-fluorescein diacetate (DCFH-DA) (D6883) (Sigma, St Louis, MO, USA) according to the manufacturer's instructions. Briefly, cells were loaded with DCFH-DA (at a final concentration of 10 μM for 30 min), washed with ice cold Hanks Balanced Salt Solution, then observed under a Zeiss LSM710 laser confocal microscope (Carl Zeiss, Germany) equipped with Zen software to process the images.
ROS assays SW480, LoVo and RKO cell lines were treated with LDL (100 μg/ml) for 36 h, followed by ROS measurement using dichloro-dihydro-fluorescein diacetate (DCFH-DA) (D6883) (Sigma, St Louis, MO, USA) according to the manufacturer's instructions. Briefly, cells were loaded with DCFH-DA (at a final concentration of 10 μM for 30 min), washed with ice cold Hanks Balanced Salt Solution, then observed under a Zeiss LSM710 laser confocal microscope (Carl Zeiss, Germany) equipped with Zen software to process the images.
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