In Vitro Assays for Measuring Intercellular Coupling Among Peripheral Circadian Oscillators.

Circadian clocks can be found in nearly all eukaryotic organisms, as well as certain bacterial strains, including commensal microbiota. Exploring intercellular coupling among cell-autonomous circadian oscillators is crucial for understanding how cellular ensembles generate and sustain coherent circadian rhythms on the tissue level, and thus, rhythmic organ functions. Here we describe a protocol for studying intercellular coupling among peripheral circadian oscillators using three-dimensional spheroid cultures in order to measure coupling strength within peripheral clock networks. We use cell spheroids to simulate in vivo tissue integrity, as well as to increase complexity of cell-cell interactions and the abundance of potential coupling factors. Circadian rhythms are monitored using live-cell imaging of spheroids equipped with circadian reporters over several days.