Real-time bioluminescence in the Nluc-WT and Nluc-TKO cells was monitored at the single cell level using an LV200 (Olympus, Japan) as described previously62 (link). EnduRen (Promega) was used as the substrate of NLuc. Values were normalized to maximum peak intensity of sample of WT cells kept in the darkness over time and to average intensity over time. To test the significance of the circadian rhythmicity and acrophase, we performed computerized analysis of normalized data in “Cosinor” and “Acro” software downloaded from the Circadian Rhythm Laboratory Software home page (http://www.circadian.org/softwar.html).
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