H2DCF-DA (Sigma-Aldrich) was used for the intracellular detection of ROS. ECFCs were seeded onto polymer and control substrates and subjected to the same photoexcitation protocol used for the in vitro tube formation assay. Immediately after the end of the protocol, cell cultures were incubated with the ROS probe for 30 min. After careful washout of the excess probe from the extracellular medium, the fluorescence of the probe was recorded (excitation/emission wavelengths, 490/520 nm; integration time, 70 ms for H2DCF-DA) with an inverted microscope (Nikon Eclipse Ti) equipped with an Analog-WDM Camera (CoolSNAP MYO, Teledyne Photometrics). To minimize the effects of the spectral overlap between the polymer absorption and emission spectra, and the probe emission, samples were turned upside down by using a homemade chamber with a 500-μm-thick channel filled with extracellular medium. Variation of fluorescence intensity was evaluated over regions of interest covering single-cell areas, and reported values represent the average over multiple cells. See figure captions for additional details about statistical analysis. Image processing was carried out with ImageJ and subsequently analyzed with Origin 8.0.
H2dcfda
Manufactured by Nikon
Sourced in Japan
H2DCFDA is a fluorescent dye used in cell biology applications. It is a cell-permeant indicator for reactive oxygen species, specifically hydrogen peroxide. The dye can be used to monitor oxidative stress in live cells.
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Lab products found in correlation
H2dcfda, by Thermo Fisher Scientific (9 mentions)
H2dcfda, by Merck Group (6 mentions)
Fluorescent microscope, by Nikon (2 mentions)
Fluorescence microscope, by Nikon (2 mentions)
Eclipse ti, by Nikon (2 mentions)
Mitosox fluorescent probe, by Thermo Fisher Scientific (1 mentions)
Fluo 4, by Thermo Fisher Scientific (1 mentions)
Fluo 4, by Nikon (1 mentions)
Lsm 600 confocal microscope, by Zeiss (1 mentions)
Ti u microscope, by Nikon (1 mentions)
Cellrox deep red, by Thermo Fisher Scientific (1 mentions)
Mitosox, by Nikon (1 mentions)
Cellrox deep red, by Nikon (1 mentions)
Mitosox, by Thermo Fisher Scientific (1 mentions)
Digital sight ds u3 camera, by Nikon (1 mentions)
Eclipse t2000 u microscope, by Nikon (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
B 2a filter block, by Nikon (1 mentions)
Amplex red hydrogen peroxide peroxidase assay kit, by Thermo Fisher Scientific (1 mentions)
Eclipse e600, by Nikon (1 mentions)
12 protocols using h2dcfda
1
Intracellular ROS Detection in ECFCs
2
Mitochondrial ROS and Calcium Sensing
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Mitochondrial O2.−, total H2O2 and free cytosolic Ca2+ levels were determined as reported before23 (link), using MitoSOX fluorescent probes (#M36008; Thermo Fisher), H2DCF-DA (#C6827; Thermo Fisher) and Fluo-4 (#F23917; Thermo Fisher). Briefly, HaCaT cells were seeded in 96 well plates and incubated up to 24 h or 7 d with sFLG in the range 0.5 to 100 µg/mL. After treatment, cells were washed twice with PBS then loaded for 30 min with the fluorescent probe (one independent probe per assay; 1 µM MitoSOX and Fluo-4; 2.5 µM H2DCFDA) and imaged with a Nikon TiU microscope (20 × objective). Pictures were analyzed and processed with ImageJ 1.53. The results show the percentage of cell signal vs. control (n = 4). Tracking experiments were performed by monitoring fluorescence levels at different times in the same cells using a Zeiss LSM-600 confocal microscope (63 × objective). Pictures were processed with Zen 2012 blue edition and ImageJ 1.53. Results show the variation of fluorescence from time 0 (> 25 cells/experiment).
3
PHMG-induced Oxidative Stress Measurement
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Cells were grown to confluence in a 12 well-plate at 24 h after seeding (5 x 105 cells/well), were treated with PHMG at the concentration of 1 μg/mL, 5 μg/mL, 10 μg/mL, and 25 μg/mL, and further incubated with 40 μM of H2DCFDA (Invitrogen, CA, USA) for 1 h. At the end of H2DCFDA incubation, cells were washed with phosphate-buffered saline (PBS), and were visualized with a fluorescent microscope (Nikon, Tokyo, Japan) [14 (link)].
4
Quantifying Oxidative Stress in Brain
5
Fluorescent Dye Staining of Fungal Cells
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H2DCFDA, CellROX DeepRed and Mitosox were purchased from Invitrogen (Molecular Probes, Eugene, OR, USA). Whole mycelia and protoplasts were incubated with dyes in the growth medium for 10–15 min and washed in the medium 3 times according to the manufacturer’s instructions. Samples were observed and photographed under a Nikon fluorescence microscope using a filter with 495 nm excitation and 500–550 nm emission wavelengths (H2DCFDA and Mitosox staining) or a filter with 510 nm excitation and 580 nm emission wavelengths (CellROX Deep Red staining).
6
Intracellular ROS Levels in DLD1 Cells
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Intracellular ROS levels after 1, 3, 6 and 24 h H.i. treatment (0.0–1.5 mg/ml) were measured through dichlorofluorescein (DCF) assay. Briefly, 20 min before the end of H.i. treatment, DLD1 cells were incubated with 10 μM 2′,7′ – dichlorodihydrofluoresceine diacetate (H2-DCFDA) (Sigma) at 37°C and 5% CO2. Cells were washed with PBS 1x, trypsinized, and measured for oxidation of H2-DCFDA by fluorescence microscopic analysis using Nikon Eclipse Ti equipped with Digital Sight camera DS U3 (Nikon, Tokyo, Japan). Fluorescence images were digitally acquired and processed for fluorescence intensity using the analysis software Image J. Mean fluorescence values were determined by averaging the fluorescence of at least 100 cells/treatment conditions. The experiment was performed in triplicate.
7
Quantification of ROS and Cell Death
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ROS quantification was performed using the fluorescent probe 2,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, λEx/Em = 495/529 nm; Thermo Fisher, Madrid, Spain). Cell death quantification was performed using the fluorescent probe propidium iodide (PI, λEx/Em = 535/617 nm; Thermo Fisher, Madrid, Spain). Brain slices were incubated with both probes for 45 min at a concentration of 10 µL/mL for H2DCFDA and 1 µL/mL for PI, in the presence of 1 µL/mL of Hoechst at 37 °C. Fluorescence from striatum slices was then recorded for H2DCFDA (λEx/Em = 495/529 nm), PI (λEx/Em = 535/617 nm), and Hoechst (λEx/Em = 350/461 nm), in an inverted Nikon Eclipse T2000-U microscope (Nikon, Tokyo, Japan). NIS-Elements BR 4.10.04 64-bit software was used to analyze the images.
8
Measuring ROS and H2O2 in Plants
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ROS accumulation was also detected using the fluorescent probe 2′,7′-dichlorofluorescin diacetate (H2DCFDA) (Sigma-Aldrich). Briefly, leaf disks were first vacuum-infiltrated (twice for 5 min) in 10 μM of H2DCFDA and observed under a Nikon Eclipse E600 epifluorescence microscope (Nikon, Tokyo, Japan) equipped with a Nikon B-2A filter block (450–490 nm excitation filter, 505 nm dichroic mirror, 520 nm barrier filter). The pixel intensities of fluorescence images, acquired using the microscope, were determined by using Image, J-software (NIH, USA).
H2O2 content was detected by the Amplex red hydrogen peroxide/peroxidase assay kit (Invitrogen).
H2O2 content was detected by the Amplex red hydrogen peroxide/peroxidase assay kit (Invitrogen).
9
Mitochondrial Activity and Oxidative Stress in Oocytes
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To assess ΔΨm, denuded MII-stage oocytes were incubated with 2 μM JC-1 (Invitrogen, Waltham, MA, USA) for 1 h at 37.5°C in the dark. The ΔΨm of oocytes was then calculated as the ratio of
red fluorescence intensity (J-aggregates; corresponding to activated mitochondria) to green fluorescence intensity (J-monomers; corresponding to inactive mitochondria) using ImageJ software.
The fluorescence intensity of the resulting oocytes was analyzed using a fluorescence microscope (Nikon). ROS levels were measured by a 2′,7′-dichlorofluorescein assay (H2DCFDA; Thermo
Fisher Scientific, Waltham, MA, USA). In brief, denuded MII-stage oocytes were cultured in 0.1% BSA-PBS containing 10 μM H2DCFDA for 15 min at 37.5°C in the dark, and then visualized at an
excitation of 485 nm and emission of 535 nm. GSH levels were quantified with the CellTracker™ Blue dye (4-chloromethyl-6, 8-difluoro-7-hydroxycoumarin, CMF2HC; Invitrogen). In brief, denuded
MII-stage oocytes were incubated in 0.1% BSA-PBS medium containing 10 μM CMF2HC for 15 min at 37.5°C in the dark, and then visualized at an excitation of 371 nm and emission of 464 nm. The
fluorescence intensity (1 sec after the shutter opening with 10 msec exposure for H2DCFDA; 3 sec after the shutter opening with 100 msec exposure for CMF2HC) of the resulting oocytes was
analyzed by fluorescence microscopy (Nikon) using ImageJ.
red fluorescence intensity (J-aggregates; corresponding to activated mitochondria) to green fluorescence intensity (J-monomers; corresponding to inactive mitochondria) using ImageJ software.
The fluorescence intensity of the resulting oocytes was analyzed using a fluorescence microscope (Nikon). ROS levels were measured by a 2′,7′-dichlorofluorescein assay (H2DCFDA; Thermo
Fisher Scientific, Waltham, MA, USA). In brief, denuded MII-stage oocytes were cultured in 0.1% BSA-PBS containing 10 μM H2DCFDA for 15 min at 37.5°C in the dark, and then visualized at an
excitation of 485 nm and emission of 535 nm. GSH levels were quantified with the CellTracker™ Blue dye (4-chloromethyl-6, 8-difluoro-7-hydroxycoumarin, CMF2HC; Invitrogen). In brief, denuded
MII-stage oocytes were incubated in 0.1% BSA-PBS medium containing 10 μM CMF2HC for 15 min at 37.5°C in the dark, and then visualized at an excitation of 371 nm and emission of 464 nm. The
fluorescence intensity (1 sec after the shutter opening with 10 msec exposure for H2DCFDA; 3 sec after the shutter opening with 100 msec exposure for CMF2HC) of the resulting oocytes was
analyzed by fluorescence microscopy (Nikon) using ImageJ.
10
Nanoparticle-Induced Oxidative Stress Assay
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Cells grown to confluence in 12 well-plate at 24 hr after seeding (1 × 103 cells in each well) were treated with 1, 10, 25, 50 and 100 μg/mL of ZnONPs for the designated time (0, 3, 6, 9, 12, and 24 hr), then incubated with 40 μM of 2,7-dichlorofluorescin diacetate (H2DCFDA) (Invitrogen, CA, USA) for 1 hr. At the end of H2DCFDA incubation, cells were washed with phosphate buffered saline (PBS), and were visualized with a fluorescent microscope (Nikon, Tokyo, Japan). Effects of AgNPs, CeONPs, TiONPs and SiONPs on ROS generation were also tested compared to those of ZnONPs.
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