Real-time Monitoring of Circadian Oscillations

For real-time monitoring of intracellular fluorescence circadian oscillations, 4 × 10⁴ Alu-egfp or IRAlu-egfp GH4C1 cells were seeded on glass-bottom 24 well plates (coated with poly- ornithine). The next day, the medium was changed with a growth medium containing 100 nM forskolin to synchronize the cellular circadian oscillators. 20 min later, the medium was changed again with a DMEM without phenol red medium containing 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 100 mM pyruvate. The next day, plates were transferred to an inverted microscope (Axiovert-200, Zeiss) equipped with an environmental chamber, the temperature of which was set to 37°C and CO₂ regulated to 5%. Egfp was excited through a 475/40 band-pass filter. An EMCCD camera (Rolera EM-C2, Q-Imaging) was used to collect emitted fluorescence at 530/50 nm. Time-lapse recordings of multiple regions in each well were realized over 96 hr with 1 picture every 30 min (Metamorph, Roper Scientific), using a 40X objective. The data were processed for tracking and measuring fluorescence fluctuations of individual cells with the CGE (Circadian Gene Expression) Plugging of ImageJ (Sage et al., 2010 (link)). Cells retained for analysis fulfilled two criteria: they were detected for at least 48 hr and their mean fluorescent level was 10% higher than mean level of the background.