Lv200

For bioluminescence recording, NIH3T3 cells (3T3-3-4, Riken Cell Bank, Japan, RRID: CVCL_1926) transfected with Bmal1-luc in a 35 mm cell culture dish were used. NIH3T3 cell line was authorized as follows: identification of animal species was done by PCR and isozyme analysis and mouse strain was identified by SSLP analysis.
For SCN imaging, adult Sik3^-/-; Per2^Luc/Luc and Sik3^+/+; Per2 ^Luc/Luc mice were used. Cellular rhythms were synchronized with 100 nM dexamethasone (DEX). Two hours after the DEX treatments, the culture medium was replaced with a recording medium, DMEM culture medium including 10 mM HEPES (pH 7.0), 10% fetal calf serum (FCS) and 0.1 mM luciferin, and circadian bioluminescence rhythm was monitored in Kronos (ATTO, Japan). For single-cell time-lapse imaging, LV 200 (Olympus, Japan) was used for SCN slices. SCN slices from adult Sik3^-/-; Per2 ^Luc/Luc and Sik3^+/+; Per2^Luc/Luc mice were prepared using a Microslicer (Dosaka Japan, 300 μm thick) and incubated on MilliCell membrane (Merck Millipore, Billerica, Massachusetts) in a 35-mm petri dish with culture medium (DMEM-F12 supplemented with B27, Thermo Fisher Scientific, Waltham, MA). Exposure time for a single image was 55 min, and time-lapse images were captured for five consecutive days. Movies were produced using MetaMorph software (Molecular Devices, Sunnyvale, CA).

Real-time bioluminescence in the Nluc-WT and Nluc-TKO cells was monitored at the single cell level using an LV200 (Olympus, Japan) as described previously^{62 (link)}. EnduRen (Promega) was used as the substrate of NLuc. Values were normalized to maximum peak intensity of sample of WT cells kept in the darkness over time and to average intensity over time. To test the significance of the circadian rhythmicity and acrophase, we performed computerized analysis of normalized data in “Cosinor” and “Acro” software downloaded from the Circadian Rhythm Laboratory Software home page (http://www.circadian.org/softwar.html).

Coronal brain slices including the SCN (300–400 μm thickness) were prepared from adult Per2^Luc mice using a vibratome^{34 (link),35 (link)}. Paired SCNs were excised from coronal brain slices and placed on a culture membrane (Millicell-CM; Merck, Germany) in a covered and sealed culture dish filled with medium containing 100 μM luciferin. Bioluminescence was measured in realtime with a photomultiplier tube (LM2400; Hamamatsu, Japan). The data sets were detrended by subtracting the 24 h running average from the raw data. To perform single neuron imaging, a luminescence microscope optimized for live cell imaging (LV200; Olympus, Japan) was used.

Paraffin-embedded sections of 5 μm thickness were kept at 60°C for 3 h in the oven and dewaxed with xylene and hydrated in gradient ethyl alcohol. The slides were subjected to microwave antigen retrieval treatment followed by endogenous peroxidase deactivation using 3% H₂O₂ for 10 min. After blocking of nonspecific binding with normal serum for 20 min, the sections were incubated with primary antibody against SIRT1 (8469, Cell Signaling Technology, Boston, USA) at 4°C overnight. The slides were then incubated with secondary antibodies at 37°C for 30 min, stained with DAB, counterstained with Mayer’s hematoxylin, and visualized by a highly sensitive inverted microscope (LV200, Olympus). Image analysis was performed by Image Pro Plus 6.0 software.

Terminal blood samples (1 mL) were processed by red blood cell lysis and then incubated overnight on a proprietary cell adhesion matrix (CAM)-coated plate (Vita-Assay TM AN6W, Vitatex, NY) in their culture medium. Following harvesting by CAM-dissociation using a proprietary CAM enzyme (Vitatex, NY), the cell suspension was pipetted into a microscopy dish (#0 cover glass, 0.085–0.115 mm, In Vitro Scientific). The imaging dish was placed in a bioluminescence microscope (LV200, Olympus) outfitted with a 100X/1.35 NA oil objective (UPLAPO00XOI3, Olympus) and a deep-cooled electron-multiplying charge-coupled device (EM-CCD; ImageEM C9100-14, Hamamatsu). The LV200 is also equipped with temperature, humidity, and CO₂ regulation for extended live cell imaging.
Brightfield and bioluminescent images were acquired using the 100X objective and 5-min exposure time for bioluminescent image acquisition.