Verapamil, Evans blue, triphenyltetrazolium chloride (TTC) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Dihydroethidium (DHE) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Laboratory (NY, United States). Primary antibodies against SIRT1, FoxO1 and SOD2 were purchased from Cell Signaling Technology (MA, United States). The antibody against acetylated-forkhead box O1 (Ac-FoxO1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Antibodies against β-actin, GAPDH were purchased from Beijing Zhongshan Goldenbridge Biotechnology Co., Ltd. (Beijing, China). The goat anti-rabbit and mouse secondary antibodies were purchased from Wuhan Boster Biotechnology Co., Ltd. (Wuhan, China). The SIRT1 inhibitor EX527 was bought from the Selleckchem (Houston, TX, United States). The kit for measuring malondialdehyde (MDA) was purchased from the Institute of Jiancheng Bioengineering (Nanjing, Jiangsu, China).
Dihydroethidium dhe
Manufactured by Beyotime
Sourced in China
Dihydroethidium (DHE) is a fluorescent probe used for the detection and quantification of superoxide anion (O2•−) in biological systems. It is a cell-permeable dye that is oxidized by superoxide to form a fluorescent product, which can be detected using fluorescence microscopy or spectroscopy.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Mitosox, by Thermo Fisher Scientific (6 mentions)
Fluorescence microscope, by Olympus (4 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Beyotime (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh da, by Merck Group (2 mentions)
Streptozotocin (stz), by Merck Group (2 mentions)
Sodium taurocholate, by Merck Group (2 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
β actin, by Proteintech (2 mentions)
Laser confocal microscope, by Leica (2 mentions)
Mitosox, by Yeasen (2 mentions)
Mito tracker green, by Beyotime (2 mentions)
Lsm 800, by Zeiss (2 mentions)
Atp assay kit, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
90 protocols using dihydroethidium dhe
1
Oxidative Stress Regulation in Cells
2
PDCA Modulates Cardiac Function and Inflammation
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2,4-Pyridine dicarboxylicacid (PDCA) was purchased from Selleck Chemicals (Shanghai, China). The antibody information was listed in Supporting Information Table S1 . High-fat diet (HFD, 21% fat plus 0.5% cholesterol) was purchased from Medicine Ltd. (Yangzhou, China). Dihydroethidium (DHE) was purchased from Beyotime Biotechnology (Shanghai, China). CDDP, DanShenDuoFenSuan (DSDF, one main ingredient of CDDP extracted from Danshen) and Xuesaitong (XST, another main ingredient of CDDP extracted from Panax notoginseng) were provided by Tasly (Tianjin, China). Brain natriuretic peptide (BNP) and N-terminal pro-BNP ELISA (NT-proBNP) kits were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). Malondialdehyde activity assay kit was purchased from Jiancheng Bioengineering Institute (Nanjing, China). Free fatty acid assay kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) ELISA kits were purchased from Quanzhou Runxin Biotechnology Co., Ltd. (Fujian, China).
3
In Vivo ROS Detection in Mouse Spinal Cord
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Dihydroethidium (DHE) (Beyotime, China) was intravenously injected into mice at a concentration of 0.05 mg DHE/g of mouse body weight. DHE can be oxidized by ROS in tissues and exhibit red fluorescence. After 30 min, mice were perfused with 4% paraformaldehyde to obtain spinal cords. DHE-labeled spinal cord sections were stored in the dark and subjected to immunofluorescence staining.
4
Fluorescent Probes for Toxoplasma Analysis
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The concentration of iron(II) ions was measured using the fluorescence probe f FerroOrange (Dojindio, Kyushu, Japan). The level of reactive oxygen species (ROS) in T. gondii was assessed using the fluorescence probe DCFH-DA (Solarbio, Beijing, China). The concentration of superoxide anion was determined using the fluorescence probe dihydroethidium (DHE, Beyotime Biotech, Beijing, China). Changes in mitochondrial membrane potential were evaluated using the fluorescence probe JC-1 (Beyotime Biotech, Beijing, China). Freshly released tachyzoites were incubated in a culture medium containing DFO or ammonium iron(II) sulfate for 1 h. After washing with PBS, the parasites were treated with FerroOrange, DCFH-DA, DHE, or JC-1, and then resuspended in 500 μL PBS following two additional washes. Finally, the stained tachyzoites were analyzed using flow cytometry or a fluorescence microplate reader [72 (link)].
5
Measurement of Intracellular ROS Levels
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The cells were seeded on six-well plates with cover glasses in each well and then treated with NAC for 2 h before treated with NEFA for 24 h. The intracellular levels of ROS were measured by loading the cells with the Dihydroethidium (DHE) (Beyotime Biotechnology, Shanghai, China) [19 (link),20 (link)]. The method was based on the DHE, it can enter cells freely through living cell membranes and oxidized by ROS to the product ethidium oxide, which can participate in chromosomal DNA and produce red fluorescence. Briefly, after the treatment, cells were stained with 10 µM DHE in serum-free DMEM-F12 for 30 min at 37 °C in the dark. Then the cells were washed three times with PBS. The fluorescence intensity was measured at 480 nm excitation and 590 nm emission using a fluorescence microscope (Zeiss LSM 700 META (Olympus, Tokyo, Japan)).
6
Kidney Injury Biomarker Assessment
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Antibodies specific for TNF-α, KIM-1, P-p53, p53 cleaved caspase-3, PCNA and β-actin were purchased from Abcam (Abcam, Cambridge, UK). F4/80+, anti-P-Smad2, Smad2, P-Smad3 and Smad3 were obtained from Cell Signaling Technology (CST, Danvers, MA). Anti-NOX4 was purchased from BiossBiotechnology (Bioss, Beijing, China). IRDye 800-conjugated secondary antibody was obtained from Li-cor Biosciences (NE, USA). Streptozotocin was obtained from Sigma-Aldrich (Sigma-Aldrich, St Louis, MO). Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Bestbio (Shanghai, China). Periodic acid Schiff (PAS), Creatinine Assay Kit and BUN Assay Kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Reactive Oxygen Species Assay (DCF Assay) Kit and Dihydroethidium (DHE) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Albumin Mouse ELISA Kit was obtained from Abcam (Abcam, Cambridge, UK).
7
Oxidative Stress and Mitochondrial Dysfunction
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Chlorine was obtained from Jinghua Gas Co., Ltd. (Changzhou, China). Pentoxifylline was provided by Sigma (St. Louis, MO). Kits for detecting the activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and oxidized glutathione (GSSG) were supplied by Nanjing Jiancheng Bio-Engineering Institute Co., Ltd. Primary antibodies against VEGF, PTEN-induced putative kinase 1 (PINK1), Parkin and cytochrome-c oxidase subunit IV (COX IV) were brought from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against occludin and E-cadherin were bought from Abcam (Cambridge, MA, USA). Antibodies against SOD 2 and Beclin 1 were bought from CUSABIO BIOTECH CO., Ltd. (Wuhan, China). Antibodies against HIF-1α, catalase (CAT), bcl-xl and β-actin were bought from Merck Millipore Technology (Burlington, MA), Proteintech Co., Ltd., (Wuhan, China), Cell Signaling Technology (Boston, USA) and Sigma (St. Louis, MO) respectively. Dihydroethidium (DHE) was purchased from Beyotime Co., Ltd. (Shanghai, China).
8
Quantifying Oocyte ROS Generation
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To determine the amount of ROS generation, GV oocytes were loaded with the oxidation-sensitive fluorescent probe dihydroethidium (DHE; Beyotime Institute of Biotechnology, Hangzhou, China) by incubation at 37 °C for 30 min in DPBS and then mounted on glass slides. Fluorescent signals were measured using a confocal microscope (Zeiss LSM 700). Photographs were analyzed using Image J software (Research Services Branch, National Institute of Mental Health, Bethesda, MD, USA) to measure brightness for each oocyte.
9
Oxidative Stress and Apoptosis Assay
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The sodium taurocholate was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The dihydroethidium (DHE) was purchased from Beyotime Institute of Biotechnology (Haimen, China). The antibody for HMGB1 (BM3965) was purchased from Boster Biological Technology, Inc. (Wuhan, China). The antibody specific for apoptosis regulator Bax (cat. No. ab32503), NOX2/gp91phox (cat. No. ab31092) and NOX4 (cat. No. ab133303) were purchased from Abcam (Cambridge, United Kingdom). The antibody for caspase-3 (cat. No. 9662S) was purchased from Cell Signaling Technology, Inc. (Danvers, MA, United States). The antibody for apoptosis regulator protein Bcl-2 (cat. No. GTX100064) was purchased from GeneTex Inc. (Irvine, CA, United States). All other chemicals used in this study were of analytical grade and were commercially available.
10
Measuring Intracellular ROS in CSFV-infected Cells
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The levels of intracellular ROS were determined by an oxidation-sensitive fluorescent probe DCFH-DA or dihydroethidium (DHE) (Beyotime). After CSFV infection and 3.1-MAVS transfection and N-acetyl-L-cysteine (NAC) (Beyotime) treatment for the specified time points, SUVECs were harvested and treated with 10 μM DCFH-DA or 2.5 μM DHE for 20 min at 37°C. H2O2 was served as a control. Cells were washed with PBS, and ROS fluorescence was detected using VICTOR X3 Multimode Plate Reader (Perkin Elmer, America). SUVECs were infected with lentivirus-MAVS-sh1 or lentivirus-shN (generated a GFP reporter) for 24 h, then the cells were infected with CSFV for an additional 36 h. The levels of intracellular ROS were detected using the fluorescent probe DHE (suitable for the cells expressing GFP).
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