Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent superoxide production by homogenates of freshly frozen LV tissue was measured with an assay based on lucigenin-enhanced chemiluminescence as described previously.12 ) The chemiluminescence signal was sampled every minute for 10 min with a microplate reader (Wallac 1420 ARVO MX/Light; Perkin-Elmer, Waltham, MA, USA), and the respective background counts were subtracted from experimental values. Superoxide production in tissue sections was examined by staining with dihydroethidium (Sigma, St. Louis, MO, USA) as described.13 ) Dihydroethidium is rapidly oxidized by superoxide to yield fluorescent ethidium, and the sections were examined with a fluorescence microscope equipped with a 585-nm long-pass filter. As a negative control, sections were incubated with superoxide dismutase (300 U/mL) before staining with dihydroethidium; such treatment prevented the generation of fluorescence signals (data not shown). The average of dihydroethidium fluorescence intensity values was calculated with the use of NIH Image software (ImageJ).
Dihydroethidium
Manufactured by Merck Group
Sourced in United States, Germany, China, Sao Tome and Principe, France
Dihydroethidium is a fluorescent probe used for the detection and quantification of reactive oxygen species (ROS) in biological systems. It is a cell-permeable dye that can be oxidized by various ROS, resulting in the formation of a fluorescent product that can be measured using spectroscopic techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Merck Group (15 mentions)
Cd45.2 apc clone 104, by BioLegend (8 mentions)
Ter119 apc cy7 clone ter 119, by BioLegend (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Image browser software, by Leica (5 mentions)
Dcfh da, by Merck Group (4 mentions)
N acetylcysteine, by Merck Group (4 mentions)
Wallac 1420 arvo mx light, by PerkinElmer (4 mentions)
Image pro plus 6, by Media Cybernetics (4 mentions)
Apocynin, by Merck Group (3 mentions)
Acetylcholine, by Merck Group (3 mentions)
Phenylephrine, by Merck Group (3 mentions)
Methylene blue, by Merck Group (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Confocal microscopy, by Leica (3 mentions)
Mitosox, by Thermo Fisher Scientific (3 mentions)
Facscalibur, by BD (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
175 protocols using dihydroethidium
1
Quantifying Superoxide Production in Cardiac Tissue
2
Antioxidant and Oxidative Stress Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apocynin (C9H10O3, A10809, Figure 1A ), protocatechuic acid (C7H6O4, 37580, Figure 1B ), acetylcholine (A6625), phenylephrine (P6126), dihydroethidium (DHE), 4,5-diaminofluorescein diacetate (DAF-2DA), 2,2-Diphenyl-1-picrylhydrazyl (DPPH, D9132), 2,2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH, 440914) (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, 238,813), Brij 35 (801,962), Tung oil (440,337), and fluorescein (F6377) were acquired from Sigma Aldrich (United States). Lucigenin (L6868) was obtained from ThermoFisher Scientific (United States). DMEM was purchased from Vitrocell (00,025, Brazil) and FBS from Gibco (12657029, South America). TBARS assay kit was acquired from Cayman Chemical (10009055, United States). The remaining salts and reagents were acquired from Sigma Aldrich.
3
Maturation Assay for P. vivax and P. cynomolgi
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The maturation assay for P. vivax was carried out as described previously65 (link),66 (link), while parasite growth was assessed by flow cytometry53 (link) (Fig. 5a ). In all, 200 μL of tightly synchronised ring-stage culture of P. cynomolgi was dispensed manually in the 96-well compound plate at a final parasitaemia of 0.5% and haematocrit adjusted to 2%. The assay plates were incubated at 37 °C for around 44 h in 5% CO2, 5% O2 and 90% N2 until mid-late schizonts stage (> 5 merozoites) was observed in the drug-free control wells via Giemsa staining. Each well was well mixed, and 20 µL was harvested into a small curved-bottom tube (Micronic) before 0.5 μL of 1 mg/mL dihydroethidium (Sigma) and 1 μL of 800 μM of Hoechst 33342 (Sigma) was added and made up to 100 μL with PBS. The tubes were incubated at RT for 20 min, and 100,000 events were acquired with an Accuri C6 (BD Biosciences, USA). The data were analysed using FlowJo software (Tree Star Inc.).
4
Yeast Cultivation and Zinc Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Diploid strains used in this work derived from the BY4743 genetic background. Yeast cells were grown at 30° in YPD medium (1% yeast extract, 2% peptone, 2% glucose). ZnCl2 was purchased from Sangon Biotech (Shanghai, China), and dihydroethidium was purchased from Sigma (Beijing, China).
5
Oxidative Stress Assessment in Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Superoxide generation was assessed by dihydroethidium (DHE, Sigma-Aldrich) staining in cultured NRVMs and freshly frozen heart tissue sections. Briefly, heart tissue sections or NRVMs were incubated with 40 μmol/l DHE for 1 h at room temperature, protected from light. The images were then obtained using a Leica laser scanning confocal microscope. Lipid peroxidation was determined by measuring malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) generation in NRVMs or heart tissues. MDA was measured using a commercial detection kit (S0131, Beyotime, Beijing, China) according to the instructions. 4-HNE content was measured using a commercially available kit (ab238538, Abcam, USA).
6
Detecting Cellular Oxidative Stress Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The presence of general reactive oxygen species (ROS) production was detected with 2,7-dichlorodihydroflourescein diacetate (DCFH-DA) (Sigma; D6883). This fluorogenic dye is widely used to measure general level of oxidative stress, as it measures hydroxyl, peroxyl and other ROS activity within the cell according to manufacturers instruction. The presence of superoxide was detected with an oxidative fluorescent dye dihydroethidium (DHE) (Sigma; D7008). Cardiac myocytes were rinsed with Dulbecco’s Phosphate Buffered Saline (D-PBS), then incubated in 100 μL of 10 μM DHE or DCFH-DA at room temperature for 60 min in a dark chamber. Then the dye solution was replaced with warm D-PBS and fluorescence intensity of each well was detected; excitation wavelength: 530 nm; emission wavelength: 620 nm in case of DHE (Csont et al., 2007 (link)) and excitation/emission at 495 nm/529 nm in case of DCFH-DA, as described (Csont et al., 2007 (link); Tao et al., 2007 (link); Kalyanaraman et al., 2012 (link); Ludke et al., 2017 (link)).
7
Detection of Superoxide Anions in DENV2-Infected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Superoxide anions were detected as described previously [17 (link)]. Briefly, a monolayer of C6/36 cells either with or without BiP/GRP78 overexpression was infected by the DENV2 at an MOI of 1 in a 6 cm Petri dish for 24 h. Cells were then washed with PBS and treated with trypsin-EDTA for 5 min. One milliliter of PBS containing 10% FBS was added to the cultured cell dishes and incubated with 10 μM dihydroethidium (Sigma-Aldrich) at 28°C in the dark for 30 min. Cells were then harvested and analyzed by a fluorescence-activated cell sorter (FACS) with excitation at 518 nm and emission at 605 nm (FACS-Calibur flow cytometer, Becton-Dickinson, Immunofluorometry Systems, Mountain View, CA, USA). One of three replicates is shown in the figures.
8
Quantifying Cardiomyocyte Oxidative Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reactive oxygen species (ROS) levels in cardiomyocytes as an indicator of oxidative stress were assessed by in situ production of superoxide anions with dihydroethidium (Sigma Aldrich). Cardiomyocytes were washed with preheated PBS (37°C) and incubated with 5 μmol/L of the fluorescent dye dihydroethidium dissolved in DMEM without FBS for 30 mins at 37°C. Fluorescent images were acquired by microscopy, and averagely 9 high power fields (×600 magnification) per group were analyzed for fluorescence intensity using the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
9
Quantification of ROS Production in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect and quantify the production of ROS, 200 μl dihydroEthidium (Sigma-Aldrich; 1 mg/ml solution) was administered via the tail vein injection51 (link) to 4 weeks post-Tam Dicerfl/fl/DATCreERT2 mice and controls as described in Andrews et al.35 (link) The mice were killed 3.5 h after injection; brains were dissected, fixed overnight in 4% PFA, processed for vibratome sections and immunostained with anti-TH antibody (Millipore). Ethidium fluorescence was visualized using a Zeiss LSM780 confocal microscope.
10
Adiponectin-Mediated Cell Signaling Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Thermo Scientific, Waltham, MA, USA. Hank's balanced salt solution (HBSS), rat recombinant globular adiponectin (gAd), Z-VAD-FMK, necrostatin-1, dihydroethidium, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide kit were purchased from Sigma-Aldrich, St. Louis, MO, USA. Antibodies against cleaved caspase-3, caspase-3, RIP1, RIP3, NF-κB, p38MAPK, phosphorylated-NF-κB, phosphorylated-p38MAPK, Bcl-2, Bax, and GAPDH as well as HRP-conjugated anti-rabbit IgG antibody were obtained from Cell Signaling Technology, Inc., Danvers, MA, USA.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!