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In brief, 20 l of Viability/Cytotoxicity Reagent containing both GF-AFC was added to each well, followed by 30 sec mixing at room temperature and incubation at 37C for 30 min

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In order to identify the optimum hirudin concentration, 2.5, 12.5, and 20 g/ml thrombin was incubated with increasing concentrations of hirudin (14 nM to 14 M) in activation buffer