Sh30031fs

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.

ChP explants were dissected in HBSS (Fisher, SH30031FS) and maintained on wet ice until plated. Only the posterior leaflet of the LV ChP was retained for analysis due to empirically determined limitations of the oxygen availability in the XFe96 Agilent Seahorse system. Tissue explants were plated on Seahorse XFe96 spheroid microplates (Agilent, 102905-100) coated with Cell TAK (Corning), in Seahorse XF Base Medium (Agilent, 102353–100) supplemented with 0.18% glucose, 1mM L-glutamine, and 1 mM pyruvate at pH7.4 and incubated for 1 h at 37 °C in a non-CO₂ incubator. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were measured via the Cell Mito Stress Test (Agilent, 103015-100) with a Seahorse XFe96Analyzer (Agilent) following the manufacturer’s protocols. Data were processed using Wave software (Agilent). ATP production was calculated as the difference in OCR measurements before and after oligomycin injection, as described by the manufacturer’s protocol (Agilent, 103015-100). Calcein AM was used to normalize between wells (Invitrogen L-3224).