Euk 8

Manufactured by Merck Group

EUK-8 is a synthetic antioxidant compound developed by Merck Group. It functions as a catalytic scavenger of reactive oxygen species. The core purpose of this product is to provide protection against oxidative stress in experimental settings.

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3 protocols using euk 8

Immunofluorescence Staining of Neural Markers

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Primary antibodies used were: mouse anti-BrdU (G3G4; DSHB), mouse anti-GFAP (Thermo Fisher Scientific), rhodamine-conjugated mouse anti-BrdU (Millipore), mouse anti-nestin (Rat-401), mouse anti-NeuN (Millipore), mouse anti–Nqo-1 (Santa Cruz Biotechnology), mouse anti-p62 (Abcam), mouse anti ubiquitin (Santa Cruz Biotechnology), mouse antivinculin (Sigma-Aldrich), rabbit anti-Atg5 (Novus Biologicals), rabbit anti-Atg16L1 (Abgent), rabbit anti-GFAP (Dako), rabbit anti-Ki67 (Spring Bioscience), rabbit anti-LC3 (Cell Signaling), rabbit anti-NBR1 (Cell Signaling), rabbit anti-Nrf2 (Abcam), rabbit anti-p62 (Enzo), rabbit anti-Sox2 (Millipore), rabbit anti-TAX1BP1 (Abgent), rabbit anti-Fip200 (ProteinTech), rabbit anti-olig2 (Millipore), rat anti-Ki67 (BioLegend), and guinea pig antidoublecortin (anti-DCX; EMD Millipore). Secondary antibodies were goat anti–rabbit IgG-FITC, goat anti–rabbit IgG–Texas red, goat anti–mouse IgG-FITC, goat anti–mouse IgG–Texas red, goat anti–mouse IgG-HRP, and goat anti–rabbit IgG-HRP (Jackson Immunology).
DHE and EUK-8 were purchased from Sigma-Aldrich. EUK-134 was purchased from Cayman Chemical. DCFDA, MitoTracker Red, and MitoTracker Green were purchased from Invitrogen.

Wang C., Chen S., Yeo S., Karsli-Uzunbas G., White E., Mizushima N., Virgin H.W, & Guan J.L. (2016). Elevated p62/SQSTM1 determines the fate of autophagy-deficient neural stem cells by increasing superoxide. The Journal of Cell Biology, 212(5), 545-560.

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Quantifying ROS in Rotavirus-infected Cholangiocytes

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Eight well chamber slides were used to quantify the amount of ROS produced in cholangiocytes following rotavirus infection. Confluent cell monolayers in chamber slides were washed twice and infected with RRV and Ro1845 at an MOI of 10 for 1 hour at 37⁰C. Cells were then washed twice, overlaid with serum-free media containing 100 μM 2’, 7’-dichlorofluorescin diacetate (DCFDA) (Invitrogen) (a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell) and incubated at 37⁰C for 2 hours. Following incubation, the cells were washed once, overlaid with phosphate buffered saline (PBS) and visualized under fluorescent microscopy (Nikon Ti Eclipse, Japan) at 495 nm. Images were quantified by mean fluorescence intensity (MFI) using Nikon Elements software. Inhibition studies included the addition of 2 mM EUK-8, a salen manganese complex (Sigma) in overlay media.

Mohanty S.K., Donnelly B., Temple H., Ortiz‐Perez A., Mowery S., Lobeck I., Dupree P., Poling H.M., McNeal M., Mourya R., Jenkins T., Bansal R., Bezerra J, & Tiao G. (2021). High Mobility Group Box 1 Release by Cholangiocytes Governs Biliary Atresia Pathogenesis and Correlates With Increases in Afflicted Infants. Hepatology (Baltimore, Md.), 74(2), 864-878.

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Evaluating Cambogin Cytotoxicity in Cells

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Where indicated, cells were pretreated with 1000 U/ml catalase (CAT, Sigma), 100 U/ml superoxide dismutase (SOD, Sigma), 50 μM EUK-8 (Sigma), 500 μM apocynin (Sigma) and/or 20 μM ML171 (Tocris) for 2 h, or transfected with NOX1 (20 nM) or Trx1 siRNA (20 nM) for 24 h, followed by the treatment with cambogin (10 μM) for 24 h. After treatment, 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT, Sigma) solution was added to the cells and the mixture was further incubated for 4 h at 37°C. After removing the medium 100 μl DMSO was added and the absorbance was measured at 570 nm, results were normalized as the percentage of control as readout of cell viability.

Shen K., Lu F., Xie J., Wu M., Cai B., Liu Y., Zhang H., Tan H., Pan Y, & Xu H. (2016). Cambogin exerts anti-proliferative and pro-apoptotic effects on breast adenocarcinoma through the induction of NADPH oxidase 1 and the alteration of mitochondrial morphology and dynamics. Oncotarget, 7(31), 50596-50611.

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