Single-cell Bioluminescence Circadian Monitoring

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 previously^{62 (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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Hirayama J., Alifu Y., Hamabe R., Yamaguchi S., Tomita J., Maruyama Y., Asaoka Y., Nakahama K.I., Tamaru T., Takamatsu K., Takamatsu N., Hattori A., Nishina S., Azuma N., Kawahara A., Kume K, & Nishina H. (2019). The clock components Period2, Cryptochrome1a, and Cryptochrome2a function in establishing light-dependent behavioral rhythms and/or total activity levels in zebrafish. Scientific Reports, 9, 196.

Publication 2019

LV200 EnduRen

Cells Circadian rhythm Darkness Promega

Corresponding Organization : The University of Tokyo

Other organizations : Nagoya City University, Yamaguchi University, Toho University, Kitasato University, National Center For Child Health and Development, University of Yamanashi

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Variable analysis

independent variables

Cell type (Nluc-WT and Nluc-TKO cells)

dependent variables

Real-time bioluminescence

control variables

Experimental setup (LV200 Olympus, Japan)
Substrate (EnduRen, Promega)
Normalization (maximum peak intensity of WT cells kept in darkness over time, average intensity over time)

controls

Positive control: WT cells kept in darkness over time
Negative control: Not explicitly mentioned

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