Fluorodish culture dishes

Live control- or rottlerin treated GFP-LC3 mPaSC grown on fluorodish culture dishes (World Precision Instrument) were imaged with an inverted Nikon Eclipse TE2000-S fluorescence microscope equipped with a CoolSnap camera (Roper Scientific). For quantification of GFP-LC3 puncta, cells displaying >10 brightly fluorescent GFP-LC3 puncta were counted as positive. For assessment of mitochondria membrane potential, treated GFP-LC3 cells were loaded with MitoTracker Red CMXRos and imaged as indicated above. Images were analyzed using the MetaMorph imaging system (Universal Imaging Corporation).

Nine adult PER2::LUC mice were killed by approved Schedule 1 UK Animal (Scientific Procedures) Act 1986 protocol. Briefly, animals were terminally anaesthetised with the intra-peritoneal injection of sodium pentobarbital (80 mg/kg) and decapitated. Brains were immediately removed and cut into 250 μm thick coronal brainstem slices containing the intermediate part of the DVC (at the level of the AP) in the ice-cold Hank’s Balances Salt Solution (HBSS, Sigma, Germany) supplemented with 1 mg/ml penicillin-streptomycin (Sigma, Germany) and 0.01 M HEPES (Sigma) using a vibroslicer (Camden Instruments, UK). Following, the DVC was manually dissected with the scalpel and placed on the 30 mm Millicell cell culture inserts (Merck, Germany) in glass coverslip sealed Fluorodish culture dishes (World Precision Instruments Ltd., USA) with sterile culture medium (Dulbecco’s Modified Eagle’s Medium; DMEM, Sigma) supplemented with 0.1 mM luciferin (Promega, USA), B27 (Gibco Invitrogen Ltd, USA), 1 mg/ml penicillin-streptomycin (Gibco Invitrogen Ltd), 10 mM HEPES (Sigma) and 3.5 g/L d-glucose (Sigma). For the AP-disconnection study, the APs were totally cut off the DVC using the surgical blade and further placed on the same culture membrane near the remaining parts of the DVC.