Lumicycle 96

Adherent islets were synchronized by a 1-h pulse of forskolin (10 μM; Sigma, Saint-Louis, Missouri, United States of America) with a subsequent medium change. The islets were subjected to continuous bioluminescence recording in CMRL medium containing 100 μM luciferin (D-luciferin 306–250, NanoLight Technology) during at least 5 consecutive days. For the experiments with ceramide and hexosylceramide biosynthesis inhibitors, 100 nM N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyl]-decanamide, monohydrochloride (PDMP, Cayman Chemical) or 100 nM myriocin (Sigma-Aldrich), respectively, were applied together with forskolin synchronization pulse and added to the recording medium for the entire experiment duration. Bioluminescence pattern was monitored by a homemade robotic device equipped with photomultiplier tube detector assemblies, allowing the recording of 24-well plates [93 (link)] or by LumiCycle 96 (Actimetrics). In order to analyze the amplitude, period length, and phase of time series without the variability of magnitudes, raw data were processed in parallel graphs by moving average with a window of 24 h [13 (link)]. Cell apoptosis was assessed where indicated with Cell Death Detection ELISA kit (Roche) in the end of bioluminescence recording experiments, according to manufacturer instructions.

ChP explants from Per2:luc mice^{43 (link)} were dissected in HBSS (Fisher, SH30031FS) and maintained on wet ice until plated. Explants were transferred to 24 well plates with 500 μL of filter-sterilized Lumicycle media (phenol-free DMEM (Sigma D-2902), 10 mM HEPES (pH 8.0), 4 mM sodium bicarbonate, 25 mM D-glucose, 1 × B27 (Gibco), 4mM L-glutamine, Penicillin/Streptomycin) freshly supplemented with 100 mM beetle D-luciferin (Promega, E1601) and/or 100 nM dexamethasone. PCR plate sealer was used to seal the wells for the duration of the experiment (ThermoFisher) and placed in a Lumicycle-96 (Actimetrics) in a water-jacketed incubator at 35˚C. Luminometry was performed by iterative measurement in 60 s bins, 10 times per hour. Luminometry data was analyzed with Lumicycle analysis software (Actimetrics). Only traces with a goodness-of-fit of at least 80% were included. Period was measured using the χ² function.