Luciferin sodium salt

β-TC6 WT, Bmal1^−/−, and Clock^−/− cells were infected with a lentivirus expressing a Per2-dLuc reporter construct (gift from A. Liu, University of Memphis) (Liu et al. 2008 (link)) and maintained in DMEM with 10% FBS and 2.5 µg/mL blastocidin (Sigma) to select for stable Per2-dLuc integration. Sealed cell cultures were incubated in 1.2 mL of DMEM containing 352.5 µg/mL sodium bicarbonate (Gibco), 10 mM HEPES (Gibco), 2 mM L-glutamine, 1% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate (Corning), and 0.1 mM luciferin sodium salt (Biosynth AG). Cultures were placed in a LumiCycle luminometer (Actimetrics), temperature was synchronized for 24 h (sine wave: 37°C–41°C–37°C–33°C–37°C), and plates were then maintained at 37°C. Bioluminescence was recorded continuously for several days.

PER2::LUC signal was monitored from SCN of mice as described previously^{10 (link)}. Briefly, SCN were excised from the hypothalamus of transgenic mice expressing PER2 fused to LUCIFERASE (PER2::LUC) and transferred to a 0.2 µm filter (Millipore) exposed to media (1.2 ml DMEM; Gibco) containing 0.1 mM luciferin sodium salt (Biosynth AG), sodium bicarbonate (352.5 μg ml⁻¹), 10 mM HEPES (Gibco), 2 mM l-glutamine, 2% B-27 serum-free supplement (Invitrogen), penicillin (25 U ml⁻¹) and streptomycin (20 μg ml⁻¹; Gibco). Dishes were sealed, maintained and monitored in a lumicycle (Actimetrics) at 37 °C as described previously^{10 (link)}.

Mice that expressed PER2:LUC were euthanized. SCN, liver, soleus, and WAT tissues were excised. Tissues were placed in a LumiCycle (Actimetrics), at 37°C, as previously described (22 (link)). For SCN, the brain was placed in a vibrotome (Leica VT1200 S), then cut into 300 μm–thick coronal slices around the SCN region. The SCN was further isolated using a sterile scalpel. The full-length soleus muscle was isolated, whereas anatomically consistent sections of the liver and mWAT were used. All isolated tissues were spread flat on a thin filter (0.2 μm, MilliporeSigma), which on its basal side was bathed in 1.2 mL of luciferin-containing media (DMEM, Gibco). The medium contained sodium bicarbonate (352.5 μg/mL), 10 mM HEPES (Gibco), 2 mM l-glutamine, 2% B-27 serum-free supplement (Invitrogen), penicillin (25 U/mL), streptomycin (Gibco, 20 μg/mL), as well as 0.1 mM luciferin sodium salt (Biosynth AG). A round coverslip was placed on top of the dishes, which were sealed with vacuum grease. Background-subtracted data were fit to a cosine curve of equation:using sum of least squares fit to quantify phase, amplitude, period, and damping rate.