Prepare enough Renilla working solution to perform the desired number of assays (100 uL working solution per assay). Dilute Aquaphile coelenterazine (2 mg/mL) in Renilla Luciferase Assay Buffer 2.0 at a ratio of 1:50. For example, add 20 uL Aquaphile™ coelenterazine stock solution to 1 mL assay buffer. For best results, working solution (assay buffer with substrate) should be prepared fresh before each use, and used within 3 hours of preparation. Renilla working solution activity is stable for up to 3 hours, but background increases up to 60% after 5 hours at room temperature.