Multicycle

The cell-based circadian assay was carried out as described previously.^{26 (link)}Bmal1-dLuc U2OS cells were cultured in a culture medium [Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 0.29 mg/mL of l-glutamine, 100 units/mL of penicillin, and 100 mg/mL of streptomycin]. The cells were suspended and seeded onto 384-well white color solid bottom plates with 20 μL (2000 cells) per well. After incubation for 2 days, 50 μL of the explant medium (DMEM supplemented with 2% B27, 10 mM HEPES, 0.38 mg/mL sodium bicarbonate, 0.29 mg/mL L-glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin, 0.1 mg/mL gentamicin, and 1 mM luciferin, pH 7.2) was added to each well, followed by the treatment of 500 nL of compounds (8 μM to 1 nM; 3-fold serial dilution series in DMSO; final 0.7% DMSO). The 384-well plates were sealed with an optically clear film. The luminescence was recorded every 100 min with a microplate reader (Infinite M200, Tecan). The period data were obtained from the luminescence rhythm by the curve fitting program CellulaRhythm^{20 (link)} or MultiCycle (Actimetrics), both of which gave similar results. Because of transient luminescence changes upon the medium change, the first 20 h data were excluded from the analysis.