Establishment of <i>in vitro</i> circadian clock formation assay system using ES cells.

<p>(A) Scheme of the method for developing circadian oscillation <i>in vitro</i> via formation of embryoid bodies (EBs). EBs were generated from 2,000 ES cells and were seeded onto low-attachment 96-well plates in differentiating medium without LIF supplementation (see Methods). Two days later, EBs were plated onto gelatin-coated tissue culture 24-well plates (putting one ES onto one well) and cultured for several weeks. Subsequently, bioluminescence in each well was monitored by using PMT-based photon counting. (B) Averaged bioluminescence traces after <i>in vitro</i> 7, 14, 21, or 32-day differentiation of ES cells carrying <i>Bmal1:luc</i> reporter (<i>left</i>, Dexamethasone reset; <i>right</i>, Forskolin reset). Data detrended by subtracting a 24-h moving average are means with standard deviation (<i>n</i> = 24).