All BALB/c nude mice were purchased from the Shanghai Sipper-BK laboratory animal Company (Shanghai, China). Briefly, a total of 4 × 106 miR-532-5p stably overexpressing or miR-NC 786-O cells were implanted hypodermically into the right oxter of 4-week-old nude mice. The tumour volume and the weight of each mouse was measured each week, and mice were killed 8 weeks after injection. Luciferase stably expressing SN12-PM6 cells with sh-miR-532-5p or sh-miR-NC (at 2 × 106, mixed with Matrigel, 1:1) was injected into the left subrenal capsule of 5-week-old male nude mice orthotopically. Primary lesions were monitored using an in vivo imaging system (IVIS) (NightOWL II, LB983, Berthold Technologies, Germany) once a week. Eight weeks after injection, animals were killed. All primary tumours and metastases isolated from mice were used for immunohistochemical staining or histological staining with haematoxylin and eosin (H&E). All animal studies were approved by the Institutional Animal Care and Use Committee of the Shanghai Tenth People’s Hospital.
Nightowl 2
Manufactured by Berthold Technologies
Sourced in Germany
The NightOWL II is a high-performance imaging system designed for advanced in vivo and in vitro applications. It features a cooled CCD camera, multiple imaging modes, and a temperature-controlled chamber to support a wide range of experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 21 ab, by R&D Systems (1 mentions)
Anti il 4 ab, by BioXCell (1 mentions)
D luciferin, by Abcam (1 mentions)
Indigo software, by Berthold Technologies (1 mentions)
Em ccd x2 camera, by Hamamatsu Photonics (1 mentions)
30g insulin syringe, by Terumo (1 mentions)
Prism 7, by GraphPad (1 mentions)
7 protocols using nightowl 2
1
Modulating miR-532-5p Regulates Tumor Growth
2
Tumor Models and Therapeutic Antibodies
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An MCA205 fibrosarcoma cell line expressing OVA protein was previously established21 (link). RMA cells, a Rauscher MuLV-induced lymphoma21 (link) was kindly provided by Dr Akira Shibuya (University of Tsukuba, Japan). Mice were inoculated subcutaneously with 8 × 105 MCA-OVA or 1 × 105 RMA, or were intravenously injected with 1 × 105 of the OVA/firefly luciferase-expressing melanoma cell line, MO4-Luc26 (link)52 (link), in the pulmonary metastatic model. Tumour size is expressed as a tumour index, the square root of (length × width), as described previously53 (link). To measure MO4 metastasis in the lung, luminescence images were analysed using NightOWL II (Berthold Technologies)26 (link). Two hundred μg of anti-IL-6R Ab, 15A7; anti-IL-6 Ab, MP5–20F3 (BioXCell); or control rat IgG Ab (Millipore) was injected 1 day before and after immunization. Daily intraperitoneal injections of 200 μg of anti-IL-4 Ab (BioXCell) and 100 μg of anti-IL-21 Ab (R&D Systems) were performed for 2 days after T-cell transfer. Two hundred μg of anti-IL-10 Ab (JES5-2A5; BioXCell) was injected every other day after tumour inoculation. For in vivo cell depletion, mice were injected with 200 μg of anti-CD4 Ab (GK1.5), anti-CD8 Ab (2.43) or anti-Gr-1 Ab (R56–86; BioXCell) 4–5 days before T-cell transfer, immunization or tumour inoculation.
3
Colon Cancer Mouse Model Experiments
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Mouse experiments were performed under the approval of the Animal Research Committee of Kumamoto University (Approval number: A 27‐069). Next, 6−8‐wk‐old BALB/c mice were purchased from Kyudo CO., Ltd., CLEA Japan, Inc., and Japan SLC, and housed under specific pathogen‐free conditions at the Center for Animal Resources and Development (CARD, Kumamoto University).
Colon26/Luc cells (4 × 106 cells/mouse) were injected intraperitoneally (ip) into BALB/c mice. In another model, Colon26/Luc cells (5 × 105 cells/mouse) were injected into the liver using a 29G needle and syringe under laparotomy. Mice anesthetized by inhalation of isoflurane were injected ip with 2.5 mg luciferin and subjected to imaging analysis using an in vivo imaging system (NightOWL II; Berthold Technologies). Cancer‐bearing mice were treated by injection ip of ES‐ML cells that were producing IFN‐β (β‐ML) or IFN‐γ (γ‐ML).
Colon26/Luc cells (4 × 106 cells/mouse) were injected intraperitoneally (ip) into BALB/c mice. In another model, Colon26/Luc cells (5 × 105 cells/mouse) were injected into the liver using a 29G needle and syringe under laparotomy. Mice anesthetized by inhalation of isoflurane were injected ip with 2.5 mg luciferin and subjected to imaging analysis using an in vivo imaging system (NightOWL II; Berthold Technologies). Cancer‐bearing mice were treated by injection ip of ES‐ML cells that were producing IFN‐β (β‐ML) or IFN‐γ (γ‐ML).
4
Orthotopic Tumor Volume and Metastasis
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In the orthotopic model, the primary tumor volume was measured every 3 days during the whole experiment using a digital caliper and calculated using the following formula: volume (mm3) = [width (mm)]2 × length (mm) × 1/2. At the end of the experiments, tumor growth and metastasis were detected using an in vivo bioluminescence imaging system (NightOWL II; Berthold Technologies GmbH, Wildbad, Germany). Fifteen minutes after D-luciferin (BioVision, Milpitas, CA, USA) injection (4 mg per mouse), luminescence signals were detected with an exposure time of 0.1 s and 4 × 4 binning. In the tail vein injection model, bioluminescence imaging was performed with an exposure time of 60 s and a 16 × 16 binning. Photon energy and tumor area were analyzed using IndiGO software (Berthold Technologies GmbH).
5
Bioluminescent Imaging of Luciferase Expression
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Plates were sprayed with 1 ml of 2.5 mM potassium luciferine (Gold Biotechnology, St. Louis, Mo., Goldbio.com , cat. no: LUCK-1) and then imaged using a Lumazone CA Automated Chemiluminescence System (Roper Bioscience), NightOwl II (Berthold), or Flumazone (Leica M205FA adapted with Hamamatsu EMCCD X2 camera). Time course analyses were taken using MetaMorph Microscopy Automation Software in a sequence of one bright-field image followed by a 3-min dark interval and then a chemiluminescence image with a 6-min exposure every 20 min for 24 hours. Luciferase expression movies were made by combining the frames, normally three frames/s, using MetaMorph Image Analysis Software. Expression was measured by selecting the region of interest (ROI) and quantifying the analog-digital units per pixel using the MetaMorph Image Analysis Software. When indicated, the luciferase measurements are referred to as the percent change with respect to its own control. The number of PBS was determined using the DR5::Luciferase reporter through the quantification of the number of sites with high expression relative to the adjacent regions along the primary root.
6
Quantifying Lymphatic Clearance Dynamics
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Lymphatic vessel drainage function was assessed by measuring the clearance over time after intradermal skin injection of the infrared probe P20D800 (Karaman et al, 2015 (link)). Mice were anesthetized with isoflurane (2%), and 3 μl of 3 μM P20D800 was injected intradermally in the ears with a 30G insulin syringe (Terumo). For mice with D7 tumors, injection was performed in the peritumoral area. The mice were then positioned in a whole-animal fluorescence imaging system (NightOWL II; Berthold Technologies), and images were acquired with the following imaging settings: λex: 745 nm, λem: 800 nm, and an exposure time of 4 s. Subsequent images were acquired of the ears at 1, 2, 3, 4, 6, and 24 h after injection. Mice were allowed to wake up and move freely between imaging time points. Fluorescence signal intensities were adjusted to baseline ear signals before injection of tracers to calculate tissue enhancement values. The fluorescence intensity values over time were fit to a one-phase exponential decay model in GraphPad Prism 7.0 software with lymphatic clearance expressed as decay constant k (expressed in h−1) or as half-life (expressed in h) using the following equations:
7
Evaluating Ovarian Cancer Metastasis Inhibition
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Mouse experiments were approved by the Animal Research Committee of Kumamoto University. Five‐ to 6‐week‐old SCID C.B‐17/Icr‐scid/scid Jcl female mice were purchased from CLEA Japan. Mice were intraperitoneally (i.p.) injected with SKOV3 cells (2 × 107 cells/mouse) expressing luciferase. After 3 or 4 days, the mice were anesthetized by i.p. injection of medetomidine, midazolam, and butorphanol. The mice underwent bioluminescence imaging to examine the extent of ovarian cancer metastasis (NightOWL II; Berthold Technologies, Bad Wildbad, Germany). After confirmation of the engraftment of the cancer, mice were divided into control and treatment groups. The mice in the treatment group were injected twice each week with iPS‐ML (without production of IFN‐β, 1 × 107 cells/mouse) or iPS‐ML/IFN‐β (1 × 107 cells/mouse). All mice underwent luminescence image analysis to evaluate the effects of the treatment. The experimental data were analyzed using Indigo analysis software.
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