In vivo bioluminescence imaging was performed using the IVIS Spectrum in vivo imaging system series 2000 (PerkinElmer). In summary, mice were anesthetized by intramuscular (IM) injection of a ketamine/xylazine solution (5:1 ratio). After IP injection of substrate D-luciferin (GoldBio), bioluminescence imaging for luciferase expression was conducted on a cryogenically cooled IVIS system using Living Image acquisition and analysis software (Xenogen). Images were acquired 10 minutes after D-luciferin administration. Bioluminescence was detected by the IVIS imager, integrated, and digitized. The region of interest from displayed images was quantified as total photon counts using Living Image 2.50 software (Xenogen).
D luciferin substrate
Manufactured by GoldBio
Sourced in United States
D-luciferin substrate is a bioluminescent compound used in various scientific applications. It serves as a substrate for the luciferase enzyme, which catalyzes a light-emitting reaction. The chemical properties and structure of D-luciferin allow it to be utilized in biological assays and imaging techniques that rely on luminescent signals.
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Lab products found in correlation
Ivis spectrum, by PerkinElmer (2 mentions)
Ivis spectrum in vivo imaging system series 2000, by PerkinElmer (1 mentions)
Living image software, by PerkinElmer (1 mentions)
Living image software 4, by PerkinElmer (1 mentions)
Ivis spectrum in vivo imaging system, by PerkinElmer (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
9 protocols using d luciferin substrate
1
In vivo Bioluminescence Imaging Protocol
2
Bioluminescent Imaging of Tumors
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Tumor cells were implanted in the omentum as described above. Local and disseminated tumor burden was monitored by bioluminescent imaging. Mice were anaesthetised with 4% isoflurane and injected i.p. with substrate d ‐luciferin (Gold BioTechnology #LUCK‐1G, St. Louis, MO, USA) at 150 mg kg−1 in Dulbecco's PBS and, after a 5‐min interval, were placed onto the warmed stage inside the light‐tight camera box of the imager (IVIS™ Spectrum; Perkin Elmer, Waltham, MA, USA) while under 2% isoflurane. Light emitted from bioluminescent cells was recorded by the IVIS® camera system. Images were quantified for tumor burden using a log‐scale colour range, and measurement of total photon counts per second (p/s) was determined using Living Image software (Perkin Elmer).
3
Bioluminescence Imaging of Tumor Perfusion
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Anesthetized mice (1 to 2% isoflurane) were imaged using an IVIS Spectrum® small animal imaging system (PerkinElmer Inc., Waltham, MA, USA), as described previously [39 (link)]. For BLI, the mice were administered substrate D-luciferin (sodium salt; Gold Biotechnology, St. Louis, MO, USA) by subcutaneous injection in the fore back neck region (80 µL of 40 mg/mL solution in 0.9% saline). Images were acquired every minute over 35 min following luciferin injection to examine time course of the emitted light. The BLI signal was quantified as the maximum flux in photons/second using Living Image Software 4.2 (http://www.perkinelmer.com (accessed on 23 August 2022)). OXi6197 was dissolved in saline (5 mg/mL) and doses in the range 5 to 65 mg/kg were administered intraperitoneally (IP) for breast cancer studies and at 15 and 35 mg/kg for kidney cancer. To assess the changes in tumor perfusion, the BLI was subsequently determined as maximum signal during time course following administration of fresh luciferin at 2, 4, (or 6) and 24 h after drug administration, and in some cases additionally at 48 and 72 h.
4
Conformational Switch Assay for Proteins
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The conformational switch assay was carried out in a 96-well plate format. The N2CN3N, the A125C/W mutant protein, or full length Firefly luciferase (150 nM final concentration) was dispensed into test buffer (1x Phosphate buffered saline (PBS), 0.05% Chaps, 0.5% DMSO) with DMSO, DTNB, or candidate compounds at various concentrations, and incubated for 30 min. Then either DMSO or NPGB was added to the mixture to a final concentration of 30 μM. The D-luciferin substrate (Gold Biotechnology, Inc) dissolved in substrate buffer composing of 1x PBS, 2 mM MgCl2, 4 mM EGTA, 4 mM ATP, 5 mM DTT was added to a final concentration of 5 μg/ml. The mixture was further incubated for 30 min. The luminescence was recorded with 5 sec integration time using a Veritas microplate luminometer.
5
In Vivo Bioluminescence and Fluorescence Imaging
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The IVIS Lumina II Bioluminescence and Fluorescence Imaging System (Caliper Life Sciences) was used for in vivo bioluminescent imaging. Mice were injected (i.p.) with 150 μg/kg body weight D-luciferin substrate (Gold Biotechnology, St. Louis, MO) and imaging was performed 10 min later (the peak time point). Images of the tumor were taken under the following settings: exposure time = 0.5 s, f/stop = 16, medium binning, field of view = 12.5 × 12.5 cm2. Living Image software was used to quantify the bioluminescent signals, reported as units of tissue radiance (photons/s/cm2/sr).
To further obtain the whole body three-dimensional (3D) images of mice to monitor tumor growth and metastasis, fluorescence Positron Emission Tomography-computed tomography (PET-CT) imaging (Perkin Elmer, Shelton, CT) was performed, and images/videos were captured using Living Image software.
To further obtain the whole body three-dimensional (3D) images of mice to monitor tumor growth and metastasis, fluorescence Positron Emission Tomography-computed tomography (PET-CT) imaging (Perkin Elmer, Shelton, CT) was performed, and images/videos were captured using Living Image software.
6
Bioluminescence Imaging for Tumor Growth
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For bioluminescence imaging (BLI) of tumor growth rate, mice were injected i.p. with 150 μL D-luciferin substrate (30 mg/mL, Gold Biotechnology), and images were acquired 5–15 minutes after injection by IVIS Spectrum In Vivo Imaging System (PerkinElmer). BLI was quantified (photon flux (p/s)) as previously described.30 (link) Imaging began on d 1 post implantation and then done weekly.
7
Split Luciferase Complementation Assay for DENV2
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All clones were generated as described previously [24 (link)]. All proteins of DENV2 were expressed in Escherichia coli strain Rosetta 2(DE3) (EMD Biosciences) and purified through a nickel-nitrilotriacetic acid (NTA) column (Qiagen) or Glutathione sepharose 4B (GE HealthCare), followed by a gel filtration 16/60 Superdex 200 column (GE HealthCare).
Split Luciferase Complementation (SLC) Assay.
The SLC assay was performed as previously described [24 (link),26 (link),57 (link)]. Briefly, MB was prepared in 11-point titrations with concentration ranging from 390 pM to 23 μM (3-fold dilution) in the SLC assay. The GST-CLuc-NS3 protein (80 nM final concentration) was dispensed into test buffer with MB, and incubated for 30 min. Then the NLuc-E66stop NS2B was added to the mixture to a final concentration of 80 nM. The D-luciferin substrate (Gold Biotechnology, Inc.) was added to a final concentration of 5 µg/ml. The reaction mixture was incubated to 2 h at room temperature and read using a ViewLux Plate Reader. IC50-SLC values were determined by fitting dose-dependent titration points to the Hill equation.
Split Luciferase Complementation (SLC) Assay.
The SLC assay was performed as previously described [24 (link),26 (link),57 (link)]. Briefly, MB was prepared in 11-point titrations with concentration ranging from 390 pM to 23 μM (3-fold dilution) in the SLC assay. The GST-CLuc-NS3 protein (80 nM final concentration) was dispensed into test buffer with MB, and incubated for 30 min. Then the NLuc-E66stop NS2B was added to the mixture to a final concentration of 80 nM. The D-luciferin substrate (Gold Biotechnology, Inc.) was added to a final concentration of 5 µg/ml. The reaction mixture was incubated to 2 h at room temperature and read using a ViewLux Plate Reader. IC50-SLC values were determined by fitting dose-dependent titration points to the Hill equation.
8
Bioluminescence Imaging of IPTG Cycles
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All mice were imaged before each cycle of IPTG gavage, immediately after each cycle of IPTG gavage, and again after 5–8 days of IPTG abstinence. The D-luciferin substrate (Goldbio, St Louis, MO) was injected intraperitoneally, at a dose of 15 μg/g of body weight. Animals were then anesthetized using isofluorane and imaging began 10 min after administration of D-luciferin. The mice were then placed in a light-tight chamber, and images were generated using a cryogenically cooled charge-coupling device camera IVIS 100 (Xenogen, Alameda, CA). Grey scale surface images of mice were collected, and the in vivo bioluminescence was represented as a pseudocolor images. The visual output represents the number of photons emitted/second/cm2 as a false color image, where the maximum is red and the minimum is dark blue.
9
In Vivo Bioluminescence and Fluorescence Imaging
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