Imageem c9100 13 em ccd camera

Cultures prepared as above were immediately transferred to the heated stage (37°C) of the bioluminescent imaging system Luminoview LV200 (Olympus, Japan) fitted with a cooled Hamamatsu ImageEM C9100‐13 EM‐CCD camera and a 20 x 0.4 NA Plan Apo objective (Olympus). Darkness was maintained throughout the recordings and exposure time was 60 minutes for OVLT and SFO and 30 minutes for SCN. Camera gain was the same for OVLT and SFO, but due to the intensity of the signal, it was lower for SCN recordings. Images were analyzed in ImageJ, using an ROI selection tool to outline the putative single cells or whole brain areas for measuring relative bioluminescence over time. Raw data were subject to a 3‐hours running average smooth and the first 12 hours of all recordings were excluded prior to analysis. Peaks and troughs of individual bioluminescence traces were determined manually. At least three peak‐to‐peak and two peak‐to‐trough measurements were used to calculate the period and amplitude, respectively. Damping rate was determined as relative to the amplitude of the peak on day 2 for baseline or day 0 of forskolin treatment. Rayleigh plots of peak phase and their corresponding r values were created using El Temps (University of Barcelona, Spain).

hESCs were either transduced with lentivirus at the stem cell stage or were differentiated for 8 days before lentiviral transduction. Cells were treated with 100 nM dexamethasone (Sigma) for 1 hour and then the medium was changed to recording medium. Recording medium for 2D cells (serum and phenol-free DMEM) (Thermo) contained 0.1 mM luciferin, 10 mM HEPES, 305 mg/L sodium bicarbonate, 50mM B-mercaptoethanol, 200 mM L-Glutamine, B27 and Insulin-transferrin-selenium (Thermo). Recording medium for 3D pellets were optimised and contained 200 mM L-Glutamine, 5 mg/mL Ascorbic-2-phosphate, Proline 4 mg/mL and ITS. Cells at 2D stages were imaged using an Alligator bioluminescence machine Cairn Research using iXon Ultra EMCCD camera. Pellets at 3D stages were immobilised in a thin layer of low melting point 1% agarose to prevent movement and imaged by the Olympus Luminoview LV200 microscope with a cooled Hamamatsu Image EM C9100-13 EM-CCD camera (Olympus).