The use and care of the experimental animals was approved by the Ethics Committee of Chongqing Medical University. Briefly, young NOD/SCID mice (6–8-weeks old, male), which were pretreated with anti-CD122 (kindly provided by Dr. Dengli Hong; ref. 27 (link)), were subjected to a sub-lethal dose irradiation (300 cGy) at the Radiation Facility of the Army Medical University, Chongqing, China. Subsequently, 3×105 T-ALL cells per mouse were tail-vein injected into the irradiated NOD/SCID mice. The injected mice were observed for survival for up to 60 days after injection. For optical imaging, mice were intraperitoneally (i.p.) injected with D-luciferin sodium salt (Gold Biotechnology, St. Louis, MO) at 100 mg/kg in 0.1 mL of PBS. Pseudo-images were obtained by superimposing the emitted light over gray-scale images of the mice.
D luciferin sodium salt
Manufactured by GoldBio
Sourced in United States, Macao
D-Luciferin sodium salt is a chemical compound commonly used in bioluminescence research. It is the substrate for the luciferase enzyme, which catalyzes a light-emitting reaction. The sodium salt form is water-soluble, making it suitable for various biological applications.
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Lab products found in correlation
Ivis lumina 3 imaging system, by PerkinElmer (2 mentions)
Xenogen ivis 200 imaging system, by PerkinElmer (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Living image software, by PerkinElmer (1 mentions)
Isoproterenol hydrochloride, by Merck Group (1 mentions)
Coelenterazine, by GoldBio (1 mentions)
Forskolin, by Merck Group (1 mentions)
5 n ethylcarboxamidoadenosine neca, by Merck Group (1 mentions)
8 bromo camp, by Santa Cruz Biotechnology (1 mentions)
H 89 dihydrochloride hydrate, by Merck Group (1 mentions)
Living image 3, by PerkinElmer (1 mentions)
Bromosulfophthalein bsp, by Merck Group (1 mentions)
Hyclone, by Thermo Fisher Scientific (1 mentions)
Ivis spectrum in vivo imaging system, by PerkinElmer (1 mentions)
Lucna 100, by GoldBio (1 mentions)
Xt 2000i automated hematology analyzer, by Sysmex (1 mentions)
20 protocols using d luciferin sodium salt
1
NOD/SCID Mouse Model of T-ALL
2
In vivo Bioluminescence Tumor Imaging
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On days one, three and eight, following the inoculation of bioluminescent tumor cells, the animals underwent in vivo imaging using the IVIS Lumina III imaging system (PerkinElmer, Waltham, MA, USA). The transfected 4T1 tumor cells express firefly luciferase which produces bioluminescence following the addition of its substrate (luciferin). In consideration of the imaging, a 15 mg/ml solution of D−luciferin sodium salt (Gold Biotechnology, St. Louis, MO, USA) in DPBS was administered i.p. in a 150 mg/kg dose. Animals were anesthetized using ketamine-xylazine three minutes following the administration of D-luciferin, and were imaged ten minutes postinjection using the following settings: auto acquisition, F/stop = 1 and Binning = 4. Data were analyzed using the Living Image software (PerkinElmer). Regions of interest (ROI) were drawn around the luminescent tumor signal automatically using identical signal thresholds. Total radiance, a calibrated unit of the luminescence (total photon flux per second), was calculated in each ROI and used for further statistical analysis.
3
Pharmacological Modulation of cAMP Signaling
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(-)-Isoproterenol hydrochloride was purchased from Sigma-Aldrich (Cat #I6504), dissolved in water/100 mM ascorbic acid to 10 mM stock, and used at 1 μM final concentration. 5′-(N-Ethylcarboxamido)adenosine (NECA) was purchased from Sigma-Aldrich (Cat #119140), solubilized in DMSO to 10 mM stock, and used at 10 μM final concentration. Forskolin was purchased from Sigma-Aldrich (Cat #F6886), dissolved in DMSO to 10 mM stock, and used at 10 μM final concentration. Shield-1 ligand for stabilization of DD-tagged proteins was purchased from Takara Bio (Cat # 632189) and added to the cell medium to 1 μM final concentration. The cell-permeable cAMP analog, 8-Bromo-cAMP, was purchased from Santa Cruz Biotechnology (Cat #sc-201564), dissolved in DMSO and used at 1 mM final concentration within 1 month of re-suspension. The CREB inhibitor compound, 666–15, was purchased from Tocris Bioscience (Cat #5661), resuspended in DMSO and used at 100 nM final. At doses higher than 100 nM, 666–15 was toxic in our cell line. H89 dihydrochloride hydrate was purchased from Sigma-Aldrich (Cat #B1427), resuspended in DMSO to 10 mM stock, and used at 10 μM final concentration. D-luciferin sodium salt (Cat #LUCNA) and coelenterazine (Cat #CZ) were purchased from GoldBio and resuspended to 100 mM in 10 mM Hepes, and 10 mM in ethanol, respectively, and stored protected from light.
4
Evaluating LDB1 Knockdown on P388D1 Tumor Growth
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Based on past experience [24 (link)] 4–6 week-old BALB/c female mice were randomly divided into two groups: sh-NC and sh-Ldb1, and each group contains seven mice. The P388D1 cells expressing firefly luciferase (P388D1.ffluc) were transfected with sh-NC or sh-LDB1 and were injected into the tail vein with 2 × 105 cells suspensions in 100ul PBS for each mice . D6/9/12 days after tail vein injection, D-luciferin sodium salt (115144-35-9, GOLDBIO, USA) was injected into the abdominal cavity of tumor-bearing mice, and small animal live imager (Berthold, Germany) scans were performed to measure the maximum standard Avg radiance uptake value of tumors in mice. Animals were anesthetized with 2% isoflurane and set for the acquisition of PET scan within 30 min. Mice were first euthanized by inhalation of CO2, and tissues, such as liver, spleen and bones were collected for subsequent experimental procedures, including paraffin block and HE staining. IHC was performed as previously described [25 (link)]. Primary antibodies against Ki67(GB12114, Servicebio, China) were used according to the manufacturer’s recommendations.
5
Bioluminescent Tumor Imaging in Mice
6
Bioluminescence Imaging of Tumor Burden
7
Intracranial Xenograft Model of Zika Virus
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A total of 50,000 luciferase-labeled GSCs (type 387 or 4121) were co-implanted with 10,000 PFU of ZIKV-LAV or RPMI 1640 intracerebrally into the right frontal lobe of a nude mouse. After implantation, bioluminescence imaging was performed using a charge-coupled-device camera (Xenogen Corp., Alameda, CA, USA) at the indicated time points as described previously (35 (link)). Briefly, 1.5 mg of the substrate d-Luciferin sodium salt (Gold Biotechnology) was administered intraperitoneally to each mouse, and images were acquired 9 min later for 60 s. To quantify the amount of light emitted from the tumor, regions of interest (ROIs) were manually defined after imaging, and the photon flux was calculated (in photons/second/square centimeter/steradian) using Living Image 3.0 (Caliper Life Sciences, Alameda, CA, USA). For experiments assessing survival, mice were monitored until the last mouse showed neurological signs. When any mouse showed neurological signs, selected mouse brains were harvested and prepared in 5-μm-thick cryosections. The cryosections were stained with hematoxylin and eosin and subjected to histological examination.
8
Xenograft Tumor Imaging in Nude Mice
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The use and care of animals was approved by the Institutional Animal Care and Use Committee of The University of Chicago (IACUC Protocol 71328). All animal experiments were performed in accordance with the guidelines and regulations stipulated in the approved protocol. Experimentally, the SKBR3 cells stably expressing firefly luciferase were infected with retroviral vectors expressing linBulg21, cirBulg21, or seed sequence SCR control. At 48 hr after infection, 5 × 106 cells per site were injected subcutaneously into nude mice at the indicated positions (n = 5 mice, female, 6–8 weeks old, Harlan Laboratories). The animals were subjected to imaging with Xenogen IVIS 200 system (Xenogen, Alameda, CA, USA) at the indicated time points. Briefly, mice were injected (intraperitoneally [i.p.]) with D-Luciferin sodium salt (Gold Biotechnology, St. Louis, MO) at 100 mg/kg in 0.1 mL PBS. The pseudo-images were obtained by superimposing the emitted light over the gray-scale photographs of the mice. Quantitative analysis was conducted with Xenogen’s Living Image software as described.55 (link), 56 (link), 57 (link) At 26 days after transplantation, the mice were euthanatized and the tumor masses were collected for histologic evaluation.
9
In Vivo Luciferase Imaging Protocol
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For the in vivo imaging of luciferase, 8–10-week-old male animals were injected with 300 mg/kg D-luciferin sodium salt (Goldbio, St. Louis, MO, USA) intraperitoneally (i.p.) and then anaesthetized with ketamine-xylazine (100 and 5 mg/kg i.p.). The fur was removed with a fine electrical shaver. Bioluminescent imaging was performed 30 min after D-luciferin administration with the IVIS Lumina III imaging system (PerkinElmer, Waltham, MA, USA) with the following settings: exposure time 5 min and, binning 4. The bioluminescent signal of the brain was quantified as a total flux (photons/s) in equal-size regions of interest (ROI) corresponding to the top of the skull. Fluorescent optical imaging of tdTomato expression was also performed with the IVIS Lumina III imaging system. For tdTomato detection, excitation filters of 500, 520, 540 and 560 nm for spectral unmixing and an emission filter of 620 nm, auto exposure and a binning of 2 were used. After imaging, animals were placed onto a heating pad and monitored until they recovered from anesthesia.
10
Bioluminescence Imaging of Osteosarcoma Xenografts
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All animal work was conducted according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC). Intramuscular injection of tumor cells was done as described.22 (link), 30 (link), 33 (link), 37 (link), 55 (link) Briefly, human osteosarcoma 143B cells stably expressing firefly luciferase were collected and resuspended in PBS for intramuscular injection (106 cells/injection) into the quadriceps of athymic nude (nu/nu) mice (5 per group, 4–6 week old, female, Harlan Laboratories, Indianapolis, IN). At two weeks post injection, animal were anesthetized with isoflurane attached to a nosecone mask within Xenogen IVIS 200 imaging system. For bioluminescence imaging, animals were injected (i.p.) with D-Luciferin sodium salt (Gold BioTechnology) at 100 mg/kg in 0.1 ml sterile saline. Pseudoimages were obtained by superimposing the emitted light over the gray-scale photographs of the animals. Quantitative analysis was done by using Xenogen's Living Image software as described.37 (link), 56 (link), 57
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