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16 protocols using lv200

1

Bioluminescence Imaging of Luciferase-Expressing Cells

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The mutated luciferase was inserted into Bam H1/Eco RI multicloning sites of pCDNA3.1 (Invitrogen), and the vector was transfected into HeLa cells by FuGene HD. The cells were cultured in DMEM containing 10% FBS overnight and subjected to bioluminescence microscopy by the addition of D-luciferin (1 mM) in the culture medium.
The bioluminescence image of the cells was captured by a luminescence microscope LV200 (Olympus, Tokyo, Japan) [11 ,12 ] equipped with UPLFLN60×OI objective lens (NA 1.25, Olympus) and DP74 color CCD camera (Olympus) or ImagEM C9100-13 EM-CCD camera (Hamamatsu Photonics, Shizuoka, Japan).
The culture medium of HeLa cells expressing luciferase was replaced with Hank's Balanced Salt Solution (Invitrogen) containing 1 mM D-luciferin. The emission spectrum of the cells was determined with the LumiFl-Spectrocapture AB-1850 at 37 °C.
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2

Circadian Dynamics of Islet Cells

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Human islets attached to glass-bottomed dishes (Willco Wells BV) were transduced with Pppg-mCherry adenovirus to label α-cells, RIP-GFP lentiviruses to label β-cells and with Per2-luc bioluminescence reporter. Islets synchronized by forskolin pulse were subjected to combined bioluminescence-fluorescence time-lapse microscopy (12 (link)) using Olympus LV200 workstation equipped with a 63× UPLSAPO objective and EM CCD camera (Image EM C9100-13, Hamamatsu). The recorded time-lapse images were analyzed on Fiji application (ImageJ), with individual cells tracked in the bioluminescence and fluorescence channels using a modified version of ImageJ plug-in CGE (27 (link)). Bioluminescence signal was measured over α- and β-cells within the encircled cell area (Fig. 2 A and B and Movies S1 and S2). Measuring of expression levels was performed on the labeled and tracked cells in the bioluminescence images over time. To assess the circadian characteristics of single-cell profiles, a JTK_Cycle fitting method was utilized (56 (link)).
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3

Bioluminescence Imaging of Live Cells

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After luciferin substrate addition, the validation setup was transferred to the stage of luminescence microscope (LV 200, Olympus). Bioluminescence was imaged with a UPLSAPO 60× oil objective (NA: 1.35) and transmitted to a cooled CCD camera. Time-lapse images were collected with 5 s acquisition times and EM gain was set at 1200× (photon imaging mode, Hamamatsu) Active cells were imaged at the central hydrogel position within a confocal volume at the maximal working distance (i.e. 150 µm) from the glass coverslip.
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4

Bioluminescent Monitoring of Circadian Rhythms

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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-/-; Per2Luc/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+/+; Per2Luc/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).
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5

Single-cell Bioluminescence Circadian Monitoring

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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 previously62 (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).
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6

Droplet Trapping and Manipulation in Microfluidic Anchors

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Droplets were formed using two separate flow focusers and droplets flowed into a 2 mm wide channel containing an array of anchors (Figure 1b). The channel height was 25 μm. The circular anchors had a diameter of 50 μm, a depth of 25 μm and were spaced 150 μm apart. The oil phase used was fluorinated oil containing surfactant, QX100 (Biorad). The aqueous droplets contained 0.1 % m/v pluronic F-68 (Affymetrix) for all experiments. Pluronic F-68 was found to reduce undesired droplet fusion. For this combination of channel depth, anchor depth and fluids, droplets would remain in the anchors for external oil flows of less than about 100 μL/min. The array could be entirely cleared of all droplets at higher external oil flows (about 200 μL/min). Fluid flow was controlled using computer-controlled syringe pumps (Nemesys, Cetoni). All images were taken with an inverted bioluminescence microscope (Olympus LV200) equipped with a light-tight enclosure and multicolor fluorescence imaging capacity.
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7

Radioluminescence and Fluorescence Imaging

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Radioluminescence imaging was performed using a bioluminescence microscope (LV200, Olympus) outfitted with a 40×/1.3 NA oil objective (UPLFLN40XO, Olympus), and a deep-cooled electron-multiplying charge-coupled device (EM-CCD; ImageEM C9100-14, Hamamatsu). All samples were imaged using 4×4 binning and an electron-multiplication gain of 1200. Fluorescence imaging was performed on Leica DM6000B microcope using a Hamamatsu C11440 fluorescence camera and a Leica DFC450 brightfield camera, with 20× magnification and an exposure time of 4 seconds.
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8

Circadian Rhythms Monitoring in Mouse SCN

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Coronal brain slices including the SCN (300–400 μm thickness) were prepared from adult Per2Luc mice using a vibratome34 (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.
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9

Immunohistochemical Analysis of SIRT1

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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.
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10

Bioluminescent Live Cell Imaging

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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.
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