Icariin (purity 98.75%) and JC-1 dye were purchased from MedChem Express (MCE, NJ, USA). The dihydroethidium (DHE) probe and mitochondria isolation kits were obtained from Beyotime Biotechnology (Jiangsu, China). The in situ cell death detection kit (Roche) was obtained from Sigma-Aldrich (St. Louis, MO, USA); and the primary antibodies against Sirt3, Sirt1, PGC-1α, Mfn2, Cyt-b, and β-actin, as well as the goat anti-rabbit and goat anti-mouse secondary antibodies were obtained from Abcam Biotechnology (Cambridge, MA, USA). The primary antibody against Apelin was obtained from Signalway Antibody (SAB, USA). Empty adenoviral vectors (Ad-EV) and recombinant adenoviral vectors expressing Apelin (Ad-Apelin), Sirt3 (Ad-Sirt3) or Apelin-specific small hairpin RNA (Ad-sh-Apelin), as well as Sirt3-specific small hairpin RNA (Ad-sh-Sirt3), were purchased from Hanbio Technology Ltd. (Shanghai, China). The titer of the adenoviruses was approximately 1.2×1010 PFU/mL.
Dihydroethidium dhe probe
Manufactured by Beyotime
Sourced in China
Dihydroethidium (DHE) is a fluorescent probe used to detect and quantify superoxide (O2-) levels in biological samples. It functions by undergoing oxidation in the presence of superoxide, resulting in the formation of ethidium, a fluorescent product. This probe allows for the assessment of oxidative stress and superoxide production in various cell types and experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
In situ cell death detection kit, by Merck Group (1 mentions)
Icariin, by MedChemExpress (1 mentions)
Jc 1 dye, by MedChemExpress (1 mentions)
Goat anti rabbit and goat anti mouse secondary antibodies, by Abcam (1 mentions)
Mitochondria isolation kit, by Beyotime (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Protease inhibitor cocktail b1400, by Selleck Chemicals (1 mentions)
Phosphatase inhibitor cocktail b15001, by Selleck Chemicals (1 mentions)
Pmsf st505, by Beyotime (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
5 protocols using dihydroethidium dhe probe
1
Apelin and Sirt3 Modulate Mitochondrial Homeostasis
2
Quantifying Cellular ROS Generation
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The dihydroethidium (DHE) probe (Beyotime, Shanghai, China) was used to measure ROS generation in H9c2 cells, as previously described [13 (link)]. Briefly, cells were adjusted to 1.5×105 cells per well and cultured in a 24-well culture plate. After hypoxic or normoxic treatment, the culture media was removed, and cells were washed three times in PBS. The DHE probe (10 μmol/L) was added to the wells for 30 min at 37°C. Photomicrographs were taken and the mean fluorescence intensity (MFI) was calculated using an Olympus BX51 microscope (Olympus, Tokyo, Japan) and ImageJ software (National Institutes of Health, Bethesda, MD, USA), respectively.
3
Quantifying Intracellular ROS Levels
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Intracellular ROS level was determined using Dihydroethidium (DHE) probe (Beyotime) according to the manufacturer’s protocols. After washing with PBS 3 times, NP cells were incubated in the medium with the DHE reagent at 37 °C for 30 mins in the dark. At least three images of the microscopic field were randomly obtained with a fluorescence microscope and then were measured using Image J software.
4
Quantifying Oxidative Stress Markers
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Intracellular H2O2 was assayed with a kit from Beyotime Biotechnology (Nanjing, China). According to the manufacturer’s instructions, the absorbance value of each sample was calibrated to a standard concentration curve to calculate the H2O2 concentration. Results are expressed as a ratio to the concentration of control cells.
Superoxide anion was detected by probe dihydroethidium (DHE) purchased from Beyotime Biotech (Nanjing, China). The cells were cultured in 96-well plates. After being treated with brucine, each group was washed twice in PBS and then loaded with DHE (10 μM) in fresh DMEM at 37 °C in dark for 30 min. After the cells were washed with PBS twice, the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The levels of superoxide anions were expressed as a ratio to the absorbance value of control cells. The images of U251 and U87 cells stained with DHE as described above were observed by fluorescence microscope (Olympus X71, Tokyo, Japan).
MDA was assayed with a kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s protocols. MDA content was expressed as a ratio of the absorbance of each prepared sample at 532 nm to that of control cells.
Superoxide anion was detected by probe dihydroethidium (DHE) purchased from Beyotime Biotech (Nanjing, China). The cells were cultured in 96-well plates. After being treated with brucine, each group was washed twice in PBS and then loaded with DHE (10 μM) in fresh DMEM at 37 °C in dark for 30 min. After the cells were washed with PBS twice, the fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The levels of superoxide anions were expressed as a ratio to the absorbance value of control cells. The images of U251 and U87 cells stained with DHE as described above were observed by fluorescence microscope (Olympus X71, Tokyo, Japan).
MDA was assayed with a kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s protocols. MDA content was expressed as a ratio of the absorbance of each prepared sample at 532 nm to that of control cells.
5
Evaluation of DON-induced Oxidative Stress
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DON (12, 13-epoxy-3, 4, 15-trihydroxytrichotec-9-en-8-one, C15H20O6, MW: 296.32, purity ≥ 99%, CAS RN: 51481-10-8) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hematoxylin and eosin staining kit (C0105), enhanced BCA protein assay kit (P0010), RIPA lysate (P0013B), and PMSF (ST505) were purchased from Beyotime Biotechnology (Shanghai, China). Phosphatase inhibitor cocktail (B15001) and protease inhibitor cocktail (B1400) were purchased from Bimake (Houston, TX, USA). Mini-reduced glutathione (GSH) assay kit (A006-2-1,96T), superoxide dismutase (SOD) assay kit (A001-3, WST-1), and catalase (CAT) assay kit (A007-1-1, Ammonium molybdate method) were purchased from Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Shanghai, China). The 4% phosphate-buffered paraformaldehyde (C1101) was purchased from Servicebio technology (Wuhan, China). The fluorescent probe dihydroethidium (DHE) was purchased from Beyotime (Haimen, China). The ELISA kit (8-OHdG) was purchased from Elabscience Biotechnology Co. Ltd. (Wuhan, China).
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