Lumicycle

BMSCs were seeded at 20,000 cells per cm² on 35-mm dishes (n = 4 per experiment) in 2 ml of alpha MEM containing 10% FBS and 1% PS and incubated at 37°C and 5% CO₂ for 4 days with a medium change. The medium was subsequently changed to 1 ml of alpha MEM containing 10% FBS, 1% PS and 10 μM forskolin and incubated for 2 h. The forskolin-treated BMSCs were washed with phosphate buffered saline (PBS) and cultured in 2 ml F12 medium containing 10% FBS, 1% PS and 1 mM luciferin. The culture dishes were then sealed with high-pressure vacuum grease (Dow Corning Corp., Midland, MI) and concealed from light until they were loaded into the automated high-throughput luminometer (LumiCycle, ActiMetrics, Wilmette, IL). The photon count per second was collected every 10 min from each dish for 5 days. All the raw data were analyzed with the average baseline photon count. The period and amplitude were determined after performing a baseline subtraction with a polynomial filter of 16 and a smoothing of 18 to produce well-defined peaks and troughs from the raw data using a proprietary software program (LumiCycle, ActiMetrics, Wilmette, IL). The “period” was measured from the peak-to-peak x-axis distance over the days while “amplitude” was taken as the peak to trough y-axis distance in photon counts per second. The period and amplitude were compared across the different days and conditions.

The bioluminescence of cultured 3 × E-box::Luc-expressing 4T1 cells was recorded using a real-time monitoring system (Lumicycle, Actimetrics, Wilmette, USA). The ALDH-positive populations of 3 × E-box::Luc-expressing 4T1 cells were cultured on spheroid forming condition, VECELL 3D inserts with 3D tumorsphere medium XF (PromoCell, Heidelberg, Germany). The 3D inserts were placed in 35 mm dishes and stimulated with 100 nM dexamethasone for synchronization of their circadian clocks. The amplitude of bioluminescence derived from 3 × E-box::Luc was calculated using the Lumicycle analysis software (Actimetrics).

Per2^Luc knock-in reporter mice were mated with NS-ZB20KO mice. These mice were kept in a standard LD cycle. Explants were prepared and cultured as described (Liu et al., 2014 (link); Yoo et al., 2004 (link)). One hour before lights off, explants were briefly prepared and immediately placed in Hank’s balanced salt solution. Then, explants were then cultured with 1.2 ml DMEM (Product No.D2902, Sigma), supplemented with 2% B27 (Product No. 17504–044, Gibco), 10 mM HEPES (pH 7.2), antibiotics (100 U/ml penicillin, 100 U/ml streptomycin, 0.1 mM luciferin (Promega), 4.5 g/l glucose, and 4.2 mM NaHCO3. SCN and liver were cultured on the Millicell culture membranes (0.4 μM, 30 mm diameter, Millipore). Bioluminescence was mounted over 10 min intervals with the LumiCycle (LumiCycle, Actimetrics) (Yamazaki et al., 2000 (link)). Date was analyzed using the LumiCycle Analysis software as described (Liu et al., 2014 (link); Wang et al., 2010 (link)).