Beetle luciferin

Bioluminescence was recorded from SCN slices prepared from P4 PER2^LUC mice housed in a standard 12:12 h LD cycle. For the preparation of slices, brains were quickly removed and chilled in Hanks’ balanced salt solution (HBSS), supplemented with 0.01 M HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 4 mM NaHCO₃. Vibratome slices (300 μm) were cut and placed on 0.4 mm membrane inserts (Millipore) in 35-mm Petri dishes (BD Biosciences) with 1-ml HEPES-buffered DMEM supplemented with 10% newborn calf serum (Invitrogen) and 0.1 mM beetle luciferin (Biosynth). Immediately after plating, slices were transduced with either the Kv4.1-targeted shRNA- or the nontargeted shRNA-expressing AAV8. Virus-containing media was removed after 3 d. After 2 weeks in culture, Petri dishes were sealed with vacuum grease and placed under photomultiplier tubes (HC135-11MOD; Hamamatsu) at 36°C in the dark. Bioluminescence was recorded in 10-min bins for at least 5 d. The period of PER2^LUC expression was determined using Chronostar and compared using a one-way ANOVA followed by a Tukey post hoc test.