0.4 mm membrane inserts

Bioluminescence was recorded from SCN slices prepared from P4 PER2^LUC mice housed in a standard 12:12 h LD cycle. For the preparation of slices, brains were quickly removed and chilled in Hanks’ balanced salt solution (HBSS), supplemented with 0.01 M HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 4 mM NaHCO₃. Vibratome slices (300 μm) were cut and placed on 0.4 mm membrane inserts (Millipore) in 35-mm Petri dishes (BD Biosciences) with 1-ml HEPES-buffered DMEM supplemented with 10% newborn calf serum (Invitrogen) and 0.1 mM beetle luciferin (Biosynth). Immediately after plating, slices were transduced with either the Kv4.1-targeted shRNA- or the nontargeted shRNA-expressing AAV8. Virus-containing media was removed after 3 d. After 2 weeks in culture, Petri dishes were sealed with vacuum grease and placed under photomultiplier tubes (HC135-11MOD; Hamamatsu) at 36°C in the dark. Bioluminescence was recorded in 10-min bins for at least 5 d. The period of PER2^LUC expression was determined using Chronostar and compared using a one-way ANOVA followed by a Tukey post hoc test.

Per2::Luc mice 14-15 weeks of age were euthanized and sections of 0.5cm of the proximal colon were collected in Hanks’ balanced salt solution supplemented with HEPES, NaHCO3, and penicillin-streptomycin. Proximal colon from untreated animals (n=5) or animals treated for 6 days with 4% DSS (n=6) were cleaned of adipose tissue and cultured on 0.4 mm membrane inserts (Millipore) in sealed 35 mm Petri dishes with 1.5 mL of DMEM supplemented with HEPES, NaHCO3, penicillin-streptomycin, B27 and beetle luciferin (Gold Biotechnologies). Bioluminescence rhythms were measured for at least 4 days with a luminometer (Actimetrics Inc) in a light-tight incubator set to 37°C, period and amplitude were calculated with LumiCycle (Actimetrics Inc) (Kloehn et al., 2016 (link); Carmona-Alcocer et al., 2018 (link)).