Levels of superoxide anion was quantified using HPLC/fluorescence assay that employs dihydroethidium as a probe as described previously.30 (link) In brief, 15 mm long aortas were incubated in Krebs-HEPES buffer (pH 7.4) at 37°C in CO2 incubator in the absence or presence of NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 3×10-4 M) for 60 min prior addition of dihydroethidium (50 μM; Molecular Probes).20 (link) After 15 min the samples were washed of dihydroethidium and incubated in Krebs-HEPES buffer for additional one hour. Then the samples were homogenized in 100% methanol and centrifuged at 10,000 rpm. Supernatants were subjected to HPLC analysis, and the pellets were dissolved in 2% sodium dodecyl sulfate for protein quantification.
Dihydroethidium
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, Netherlands, Canada, Australia
Dihydroethidium is a fluorescent dye used in biological research. It is a cell-permeable compound that can be used to detect the presence of superoxide, a reactive oxygen species, in living cells. Dihydroethidium is oxidized by superoxide to form a fluorescent product, which can be detected using fluorescence microscopy or flow cytometry techniques.
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Lab products found in correlation
Mitosox red, by Thermo Fisher Scientific (10 mentions)
Mitosox, by Thermo Fisher Scientific (9 mentions)
Daf fm diacetate, by Thermo Fisher Scientific (6 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (6 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Trypsin, by Thermo Fisher Scientific (4 mentions)
Cm1950, by Leica (3 mentions)
Lsm 510 meta confocal microscope, by Zeiss (3 mentions)
Accuri c6, by BD (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (3 mentions)
Cell culture reagents, by GE Healthcare (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Cellquest software, by BD (2 mentions)
Z vad fmk, by Merck Group (2 mentions)
Dnase, by Merck Group (2 mentions)
222 protocols using dihydroethidium
1
Quantifying Superoxide Anion Levels
2
Quantification of Superoxide Levels
3
Measuring Spermatozoa Cellular ROS
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Total cellular ROS generation was measured using dihydroethidium (DHE; Molecular Probes, Eugene, OR, USA). Spermatozoa were incubated with 2 μM dihydroethidium (DHE; Molecular Probes, Eugene, OR, USA) and 5 nM SyG vitality stain (Molecular Probes, Eugene, OR, USA) for 15 min at 37 °C. This step was followed by a single centrifugation and resuspension in BWW. The samples were assessed via flow cytometry, and the results were expressed as the percentage of live cells that were DHE positive. As a positive control, a sperm sample was treated with 50 μM arachidonic acid (AA), while a snap-frozen sperm sample was used as a positive SyG control [26 ].
4
Medin Effects on Endothelial Nitric Oxide
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Human umbilical vein endothelial cells (HUVECs; Lonza, Walkersville, MD; passages 4–10) were exposed to medin (0.1–5 μM in 20 mM sodium phosphate, 20 mM NaCl, pH 7.4) for 20–24 h. NO gas was measured with the use of a Sievers NO analyzer (GE Analytical Instruments, Boulder, CO) and normalized to the cell count. In separate experiments, HUVECs were treated for 60 min with medin (5 μM ± pegylated superoxide dismutase (PEGSOD, 300 U/mL)), as well as untreated media control. After 45 min, acetylcholine (10−4 M) was added to the treatment media and incubated for the remaining 15 min. After treatment, the cells were washed with cold PBS, fixed with 4% paraformaldehyde in PBS and cold 100% methanol, and then washed again and stained in 5 μM dihydroethidium (Molecular Probes, Eugene, OR). Coverslips were attached to slides with SlowFade gold antifade reagent (Thermo Fisher Scientific, Waltham, MA) and imaged on an EVOS FL Auto imaging system (Life Technologies, Eugene, OR) using an RFP light cube (excitation 531/40, emission 593/40). Images were analyzed using ImageJ Java-based image processing and analysis software (NIH, Bethesda, MD).
5
Quantifying Intracellular Oxidative Stress
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Intracellular superoxide and hydrogen peroxide levels were measured using dihydroethidium [12 (link)] (HE) (Molecular Probes/Invitrogen, Carlsbad, CA, USA) and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate [13 (link)], acetyl ester (DCF-DA) (Molecular Probes/Invitrogen, Carlsbad, CA, USA), respectively. Briefly, 5 × 105 PBMCs were resuspended in 1 mL PBS containing 10 µM HE or 1 mL RPMI containing 10 µM DCF and incubated for 30 min at 37 °C in the dark. Cells were then washed twice with PBS and read on the FL-1 (for hydrogen peroxide) or FL-3 (for superoxide) channel of a Becton Dickinson fluorescence-activated cell sorter (FACS Caliber; Becton-Dickinson, Franklin Lakes, NJ, USA). Jurkat cells (5 × 105) were treated with 2 mM hydrogen peroxide (Sigma, St. Louis, MO, USA) for 15 min and stained as above as a positive control for ROS staining. Results were analyzed using Cell Quest Software (Becton Dickinson). Mean fluorescence for each sample was calculated by subtracting baseline fluorescence (unstained cells) then normalizing to the cell number.
6
Cryosection Imaging of Oxidative Stress
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Hearts were quickly removed and frozen in medium for cryosectioning. Frozen tissue blocks were transversely cut into 30 μm thick sections and slides transferred to a recording chamber. Slides were pre-incubated with warmed Tyrode solution for 15 min and then, the sections were loaded with 5 μM of dihydroethidium, a fluorescent cell-permeable dye (DHE; Molecular Probes) for 30 min (Erickson et al., 2008 (link)). DHE staining with light fixation was adapted from a published method (Owusu-Ansah et al., 2008 (link)). Images were recorded using a fluorescence microscope (Ci-E, Nikon, Japan) and the fluorescence intensity was measured using ImageJ software (NIH).
7
Multimodal Analysis of Cellular Responses
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OCR, ROS and cell apoptosis were detected by flow cytometry analysis. For evaluation of OCR, oxygen-sensitive fluorescent probe Mito-Xpress (Luxcel Bioscience, Cork, Ireland) was used to analyze the oxygen consumption of cells. For measurement of apoptotic rate, Annexin V-FITC and propidium iodide (BD Pharmigen, San Diego, CA, USA) were used to calculate the Annexin V-positive cells. For the detection of cellular ROS, cells were stained with dihydroethidium (Molecular Probes) according to the manufacturer’s instruction.
8
Intracellular ROS Quantification in Liver
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Intracellular levels of ROS, including O2−, were measured by means of the oxidation-sensitive fluorescent probe dye dihydroethidium (DHE; Ex/Em = 518/605 nm; Invitrogen/Molecular Probes). Liver tissue sections were washed in phosphate-buffered saline (PBS) and incubated with 20 μM DHE at 37 °C for 30 min. For quantitative determination, tissue sections were scanned under a microscope with a 20× objective. The cell area was quantified via DHE staining using Zeiss LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany) with subtraction of background fluorescence.
9
Measuring Oxidative Stress in HUVECs
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HUVECs were incubated with 10 µM dihydroethidium (Molecular Probes) for 30 min in a dark chamber. After three washes with HEPES buffer (10 mM HEPES, 10 mM glucose, 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 5 mM NaHCO3, 0.6 mM Na2HPO4, 1.2 mM Na2SO4, pH 7.4), images were acquired using a fluorescence microscope (BZ-8100, Keyence, Osaka, Japan).
10
Apoptosis and Oxidative Stress Assays
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We obtained an In Situ Cell Death Detection Kit (Fluorescein), thiazolyl blue tetrazolium bromide (MTT), indomethacin, quercetin, Dulbecco’s modified Eagle’s medium (DMEM), DNase, and other reagents and chemicals from Sigma–Aldrich, Merck KGaA (Darmstadt, Germany). We obtained Muse Caspase-3/9 Assay Kits and the pan-caspase inhibitor Z-VAD-FMK from Millipore, Merck KGaA (Darmstadt, Germany). We obtained dihydroethidium, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)], and MitoSOX from Molecular Probes, Thermo Fisher Scientific (Waltham, MA, USA). We purchased L-glutamine, fetal bovine serum (FBS), trypsin-EDTA, and penicillin/streptomycin from HyClone, GE Healthcare Life Sciences (Logan, UT, USA).
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