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After aspiration and washing, 100 l of substrate solution was added to each well and incubated for 30 min at room temperature. Then, 100 L of a stop solution was added, and color changes were measured at 450 nm using a BioTek ELISA reader

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Nitric oxide synthase regulates vascular tone, and tissue hypoxia during ischemia impacts both endothelial cells and smooth muscle cells, contributing to vascular dysfunction