For bioluminescent recording, SCN slices were transferred into 1.2 mL modified air medium. This was made as previously described (Hastings et al., 2005 (link)) and modified to be serum-free, corticosterone-free, and insulin-free NS21 (Bicarbonate-free DMEM, 5 mg/mL glucose, 0.35 mg/mL sodium bicarbonate, 0.01M HEPES, 2 μg/mL pen/strep, 1% glutamax, 6% corticosterone and insulin-free NS21, 0.3 mM d-luciferin). All other tissue slices were cultured and recorded in 1.2 mL low glucose (5.5 mM) DMEM (D5921), supplemented with 1% glutamax, 2% (corticosterone-free and insulin-free) NS21 and 1 mM d-luciferin. Slices were placed in a 35mm polystyrene Petri dish and sealed with a coverslip and vacuum grease. SCN slices were treated with 1 μM TTX citrate and 600 nM insulin. Whole SCN slice bioluminescence was recorded using Hamamatsu photomultiplier tube assemblies housed within a light-tight 37°C incubator, while whole tissue slices were imaged using an ALLIGATOR. Time-lapse imaging of bioluminescent SCN slices was performed using an LV200 Luminoview (Olympus). Bioluminescent images were acquired over a 30 min interval using a C9100-13 EM-CCD camera (Hamamatsu).
Luminoview lv200
Manufactured by Olympus
Sourced in Japan
The Luminoview LV200 is a laboratory imaging system designed for visualizing and analyzing luminescence-based samples. It provides high-sensitivity detection and quantification of light-emitting specimens, such as fluorescent or bioluminescent samples. The LV200 system is equipped with advanced optical components and a sensitive camera to capture detailed images and data.
Automatically generated - may contain errors
Lab products found in correlation
C9100 13 em ccd camera, by Hamamatsu Photonics (1 mentions)
Imageem c9100 13 em ccd camera, by Olympus (1 mentions)
Uplsapo, by Olympus (1 mentions)
Imagem, by Hamamatsu Photonics (1 mentions)
D luciferin potassium salt, by Fujifilm (1 mentions)
Polyethyleneimine max mw 40 000, by Polysciences (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
35 mm glass bottom dish, by AGC Techno Glass (1 mentions)
Uplanfln 40 na1.30 oil, by Olympus (1 mentions)
Dp70 colour ccd camera, by Olympus (1 mentions)
Imagem emccd camera, by Hamamatsu Photonics (1 mentions)
9 protocols using luminoview lv200
1
Bioluminescent Recording of SCN Slices
2
Time-lapse Bioluminescence Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For bioluminescence imaging, the medium was replaced with a medium containing 0.2-mM luciferin with or without 100-nM dexamethasone (DEX). Next, via the use of a bioluminescence microscope imaging system (LV200 LUMINOVIEW; Olympus Corporation), 20-minute-exposure time-lapse images of the cells were collected at 60-minute intervals.
3
Bioluminescence Imaging of Circadian Rhythms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultures prepared as above were immediately transferred to the heated stage (37°C) of the bioluminescent imaging system Luminoview LV200 (Olympus, Japan) fitted with a cooled Hamamatsu ImageEM C9100‐13 EM‐CCD camera and a 20 x 0.4 NA Plan Apo objective (Olympus). Darkness was maintained throughout the recordings and exposure time was 60 minutes for OVLT and SFO and 30 minutes for SCN. Camera gain was the same for OVLT and SFO, but due to the intensity of the signal, it was lower for SCN recordings. Images were analyzed in ImageJ, using an ROI selection tool to outline the putative single cells or whole brain areas for measuring relative bioluminescence over time. Raw data were subject to a 3‐hours running average smooth and the first 12 hours of all recordings were excluded prior to analysis. Peaks and troughs of individual bioluminescence traces were determined manually. At least three peak‐to‐peak and two peak‐to‐trough measurements were used to calculate the period and amplitude, respectively. Damping rate was determined as relative to the amplitude of the peak on day 2 for baseline or day 0 of forskolin treatment. Rayleigh plots of peak phase and their corresponding r values were created using El Temps (University of Barcelona, Spain).
4
Bioluminescence Imaging of GUVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were obtained with a Luminoview LV200 (Olympus) bioluminescence imaging system, equipped with an EM-CCD camera (0.688 MHz EM-CCD CAM-ImageEM X2, Hamamatsu, Japan). GUVs were prepared as described above with POPC, 1% DiD and 0.6 U per mL encapsulate HRP. 50 μL of GUV solution was added into an 18-well ibidi glass bottom μ-slide, which was precoated with 3% w/v BSA. After addition of 200 μM luminol and 500 μM H2O2 and the position the GUVs was determined in the bright field channel. The GUVs were monitored in the chemiluminescence and DiD channels, where the bioluminescence was monitored using a 400–500 nm filter and an exposure time of 10 min with an EM gain of 400, using a UPLSAPO 60× Apochromat objective, followed by 10 ms exposure to image the membrane dye DiD. This imaging sequence was repeated three times to measure the chemiluminescence between 0–10, 10–20 and 20–30 min.
5
Long-term bioluminescence imaging of cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bioluminescent imaging was carried out as reported previously (Ono et al.36 (link)). Before the imaging was started, the culture medium was exchanged to that containing 5% FBS, 2% B27 supplement, 2.5 mM L-glutamine, 15 mM HEPES, 0.1 mM D-luciferin potassium and 1% penicillin/streptomycin in DMEM/Nutrient Mixture F12. Bioluminescence from each cell was captured by Luminoview (LV200, Olympus) equipped with an EMCCD camera (512 × 512 pixels; ImagEM, Hamamatsu Photonics or iXon Ultra 897, Andor Tech.) cooled at −80 °C, dry objectives (40×, 0.9 NA, UPLSAPO; Olympus) and 0.2× relay lens. Imaging area is 1024 × 1024 μm2 which covers one or two islands at same time in the collagen spray method. The measurement was done every 60 min with an exposure time of 59 min for at least consecutive 7 days. In a single experiment, bioluminescent measurement was performed in 1 dish. During measurement, the culture medium was not exchanged.
6
Bioluminescence Imaging using Luciferase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bioluminescence by luciferase was observed using Luminoview LV200 (Olympus, Osaka, Japan). Luminescence was acquired in MetaMorph® imaging system (Molecular Devices, Sunnyvale, CA). Pv11 cells were attached onto a 35 mm glass bottom dish (AGC Techno Glass Co., Ltd., Tokyo, Japan) using 50 µg/ml cationic polymer, Polyethyleneimine “Max” MW 40000 (Polysciences, Inc., Warrington, PA). The cationic polymer was prepared as described previously41 (link). D-luciferin potassium salt (Wako Pure Chemical Industries, Osaka, Japan) was dissolved in distilled water and kept at 4 °C in the dark until use.
7
Whole-Brain Imaging with EM-CCD Camera
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Images were taken with the Olympus Luminoview LV200 (Olympus, Japan) fitted with a cooled Hamamatsu ImageEM C900-13 EM-CCD camera and a NA Plan Apo objective (Olympus, Japan) on a heated stage kept at 37 °C. Gain and exposure time was constant throughout the recordings. For these experiments we used 30 min exposure time, but to match it with our previous data30 (link), we further binned the datapoints into 1 h epochs. Raw images were initially analysed in FIJI (ImageJ, NIH, USA) using a polygon region of interest tool for the whole brain areas.
8
Inverted Bioluminescence Microscopy Setup
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Figure 1 shows the inverted bioluminescence microscope used in our studies (Luminoview LV200; Olympus, Tokyo, Japan). Bioluminescence emitted from live cells in a culture dish was collected by an objective lens and the light passed through an imaging lens that then transmitted the image to a CCD camera. The objective lenses used in this study were UPlanFLN 40×/NA 1.30 Oil and UPlanFLN 100×/NA 1.30 Oil (Olympus). The DP70 colour CCD camera (Olympus) and ImagEM EM-CCD camera (C9100–13; Hamamatsu Photonics, Shizuoka, Japan) were equipped for an LV200 microscope. The imaging lens was developed with a focal length of 36 mm with an NA of 0.2. A stage-top incubator with temperature and CO2 gas controllers (MI-IBC-IF; Tokai Hit Co., Shizuoka, Japan) was added to the sample stage. The observation area was covered with a dark box.
9
Imaging of Cellular Calcium Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were treated with 5-μM 11-cis-retinal in the medium for at least 60 min, and rinsed twice with Basal Salt Solution. Cells were loaded with Fluo-4 AM (5 μM) for fluorescence imaging or soaked in Basal Salt Solution supplemented with 2 mM d -luciferin for bioluminescence imaging in the dark room. The cells were allowed to stabilize for 60 min (Fluo-4) or 30 min (d -luciferin) prior to the experiment. The culture dish was placed on the stage of a microscopic luminescence imaging system (LuminoView LV200; Olympus), and cells were observed with a 40× objective and maintained at room temperature (~24°C) during the experiments. Fluo-4 was excited by light pulses (500 msec; 0.05 μW/mm2) through 470–490 nm band-pass filters from 100-W halogen bulb. The stimulation light was guided to the microscope stage, as described in Fluorescence Ca2+ imaging section.
Fluorescence and bioluminescence images were collected using a cooled electron-multiplying charge-coupled device camera (iXon; Andor Technology plc; Belfast, UK). Images were analysed using the MetaMorph software. Each cell was chosen as an ROI, and its fluorescence or luminescence intensity at each time point was measured.
Fluorescence and bioluminescence images were collected using a cooled electron-multiplying charge-coupled device camera (iXon; Andor Technology plc; Belfast, UK). Images were analysed using the MetaMorph software. Each cell was chosen as an ROI, and its fluorescence or luminescence intensity at each time point was measured.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!