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2 protocols using 3 benzyl 7 2 ben zoxazolyl thio 1 2 3 triazolo 4 5 d pyrimidine vas2870

1

Apoptosis Induction in Cell Cultures

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Bovine serum albumin (BSA), Fetal bovine serum (FBS), L-15 Leibowitz medium, Dulbecco’s modified Eagle medium and Ham’s nutrient mixture F-12 (DMEM/F12), Penicillin (10,000 IU/ml), Streptomycin (10,000 mg/ml), Dulbecco’s phosphate buffered saline (PBS) were supplied from Gibco BRL, Life Technologies (Paisley, Scotland). Staurosporine, 3-benzyl-7-(2-ben-zoxazolyl) thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870), 3,3’-dihexyloxacarbo-cyanide iodide (DiOC6(3)), and 2-7-dichlorodihydrofluorescin di-acetate (DCFH-DA) were purchased from Sigma Chemicals (St. Louis, Missouri, USA). The pan-caspase inhibitor (z-vad-fmk) was purchased from R&D Systems (Minneapolis, Minnesota, USA). 7-Aminoactinomycin D (7-AAD) staining dye for flow cytometry was purchased from Beckman-Coulter, Inc. (Brea, California, USA). Bio-Rad protein assay kit was purchased from Bio-Rad Laboratories, München, Germany. 1,2-diaminocyclohexanetetraacetic acid (DCTA), dithiothreitol (DTT), Triton X-100, Tris-phosphate, and glycerol were supplied from Merck KGaA, Darmstadt, Germany. ATPlite 1-step assay system was supplied from Perkin Elmer, Boston, Massachusetts, USA. Caspase-Glo 3/7 Assay was purchased from Promega, Madison, Wisconsin, USA.
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2

Vascular Function Assessment with Inhibitors

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Vessels were incubated with various inhibitors for 30 minutes prior to conducting PE, ACh or SNP response curves. NO synthase inhibitor, NW-nitro-L-arginine methyl ester (L-NAME 100 μM; Sigma Aldrich), NADPH oxidase was inhibited by a non-specific inhibitor (Apocynin 100 μM) and a specific and novel inhibitor, 3-Benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine (VAS2870 10 μM; both from Sigma Aldrich St. Louis, MO). 1H-[2 (link),12 (link),55 (link)] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 10 μM; Sigma Aldrich St. Louis, MO), a guanylyl cyclase inhibitor, was also incubated with some vessels for 30 minutes prior to usage of PE, ACh or SNP. To protect against the potential deleterious effects of ROS, separate vessels, ex vivo, were incubated for 30 minutes with an antioxidant, N-acetyl cysteine (NAC 20 mM; Sigma Aldrich, St. Louis, MO), and the above myograph procedure was conducted as described above.
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