To visualize tissues expressing Luciferase (Luc) in vivo, gene construct Anc80L65.Luc and AAV9.Luc was injected intramyocardial or intracoronary. At day one, three, seven and six weeks after gene transfer the d-luciferin substrate (Biotium, Hayward, CA) was injected intraperitoneally, at a dose of 150 μg/g of body weight. Details are available in Appendix E1 .
D luciferin substrate
Manufactured by Biotium
D-luciferin is a substrate used in bioluminescence assays. It is a light-emitting compound that produces luminescence when it reacts with the enzyme luciferase in the presence of ATP and oxygen.
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7 protocols using d luciferin substrate
1
In Vivo Luciferase Imaging
2
Bioluminescent Imaging of Luciferase Activity
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The mice were anesthetized with 2% isofluorane and oxygen. The D-luciferin substrate (Biotium, Hayward, CA) was injected intraperitoneally (3.3 μg per mouse). After 10 min, 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), recording bioluminescence at 1, 10, 60 and 100 sec. The visual output represents the average radiance as the number of photons emitted/second/cm2 as a false color image where the maximum is red and the minimum is dark blue. All animals were imaged on a schedule of 3, 7, 10 and 14 days after AAV vector injection. At each time-point a “region of interest” was designated surrounding each animal in order to quantify the radiance (photons/sec/cm2/radian) being released by luciferase activity. This region was kept the same for each mouse and at each time point. The mean and standard deviation of radiance measurements was determined for each mouse group at each time point.
3
Non-Invasive Bioluminescence Imaging
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The mice were again anesthetized with 2% isoflurane and oxygen. In experiments in which AAV-fLuc was used, the d-luciferin substrate (Biotium, Hayward, CA) was injected intraperitoneally at a dose of 150 μg/g of body weight. In contrast, when AAV-gLuc was used, either 100 μg of standard coelenterazine or 100 μg of coelenterazine-SOL (Nanolight, Pinetop, AZ) was injected intraperitoneally. The mice were then placed in a dark chamber inside of a Bruker In Vivo Xtreme system (Billerica, Massachusetts). Images were generated using a cryogenically cooled charge-coupling device camera IVIS 100 (Xenogen, Alameda, CA). For each mouse, bioluminescence images were taken at either 20 min (fLuc) or 1 min (gLuc) following substrate injection. Both light and dark images of mice were collected, and the dark images were then pseudocolored using the Bruker software (Molecular Imaging software v7.5). Visual output was set to represent the number of photons emitted/second/cm2 as a false color image, where maximum intensity is colored white and the minimum is colored violet. All animals were imaged on a schedule of 3, 7, 14, 21, 28, 35, 42, and 56 days following injection of AAV into the caudal IVD.
4
Cytokine-Induced AQP3 Promoter Regulation
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The AQP3 promoter constructs generated above were used for elucidation of AQP3 promoter elements involved in the cytokine‐induced decrease in AQP3 expression. Cells were passaged and washed twice in antibiotic‐free media, followed by seeding at a density of 2.0 × 106 cells per well in a 12‐well plate. Immediately following seeding, 100 μL of premixed OptiMEM Media (Gibco, Grand Island, NY) containing 2 μg of plasmid DNA and 6 μL of TransIT‐LT1 transfection agent (Mirus Bio, Madison, WI) were added dropwise to each well. Cells were allowed to adhere to the base of the well for 5 h, followed by switch to serum‐free media for 1 h and subsequent treatment with cytokine for 12 h in serum‐free media. Cells were washed twice in PBS and collected by scraping in 250 μL of 1X lysis buffer supplied with the Firefly luciferase assay kit (Biotium, Hayward, CA). Twenty microliter of lysate was added per reaction and luminosity generated from D‐luciferin substrate (Biotium) was assessed over a 10 sec period. Data were normalized to the untreated, full AQP3 promoter construct (−1680).
5
In Vivo Bioluminescence Imaging of Mice
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At day 42 after vector injection, mice were anaesthetized with pentobarbital sodium (1%, 40mg/kg mice) via intraperitoneal injection. The D-luciferin substrate (Biotium, Hayward, CA) was injected intraperitoneally at a dose of 150 μg/g. The mice were then placed in a light-tight chamber, and images were generated using a Bioanalytical Instruments (Berthold Technologies, DE). Light was monitored in all of the experiments described at 8-12 minutes after the substrate injection. 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.
6
Bioluminescent Imaging of Luciferase Activity
7
In Vivo Bioluminescence Imaging
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After the last blood collection, mice were anaesthetized by pentobarbital sodium (1 %, 40 mg/kg mice) via intraperitoneal injection. The D-luciferin substrate (Biotium, Hayward, CA) was injected intraperitoneally at a dose of 150 μg/g. The mice were then placed in a light-tight chamber, and images were generated using a Bioanalytical Instruments (Berthold Technologies, DE). Light was monitored in all of the experiments described at 8–12 min after the substrate injection. The visual output represents the number of photons emitted/second as a false color image where the maximum is red and the minimum is dark blue.
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