Per2::LUC mice were euthanized by CO2 aspiration 3 to 5 hours before lights off in a 14-hour/10-hour light/dark cycle, and tissues were immediately dissected in a cold HBSS solution (Life Technologies). The Iris-CB complex with supporting tissues (i.e., a small rim of associated sclera) was cultured on cell culture inserts (PICM0RG50; Millipore, Burlington, MA, USA) in sealed dishes with Dulbecco's modified Eagle's medium containing a B-27 supplement (Life Technologies), 352.5 μg/ml sodium bicarbonate, 10 mM HEPES (Life Technologies), 25 U/ml penicillin, 25 μg/ml streptomycin (Life Technologies), and 0.1 mM luciferin potassium salt (Biosynth, Staad, Switzerland). All tissue cultures were incubated at 37°C, and their bioluminescence were recorded for 1 minute at 7.5-minute intervals by using a Lumicycle luminometer (Actimetrics, Wilmette, IL, USA). The overall recording period was at least 8 days. The obtained bioluminescence was detrended by using a polynomial fit line to eliminate a steady decline of background bioluminescence. The period of oscillation was decided by best-fit sine wave analysis in the Lumicycle Analysis software.