In total, 25 eight-week-old female BALB/c AnN.Cg-Foxnlnu/CrlNarl mice were purchased from the National Laboratory Animal Center, Taipei, Taiwan. One week later, 1 × 105 GBM8401-Luc tumor cells were implanted into the right cerebral hemisphere of the mice. The animals were randomly divided into four groups: shLuc, shLuc + Temozolomide (TMZ, MedChem Express, NJ, USA), shKDELC2, and shKDELC2 + TMZ treatment groups. TMZ was administered through oral gavage at 5 mg/kg/day for 7 days. The region of interest (ROI) was monitored using a noninvasive in vivo imaging system (IVIS) (Perkin Elmer, Massachusetts, USA) and measured at 0, 2, 5, and 7 days of TMZ administration. The bioluminescence intensity was compared after intraperitoneal injection of D-luciferin firefly and potassium salt (Biosynth, Thal, Swiss) in PBS. At 7 days, the mice were euthanized with tiletamine-zolazepam and xylazine, and the brains were fixed in 10% formalin, embedded in paraffin, and cut into serial sections. Animal experiments were approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taiwan (Approval number: IACUC-20-106, Date: 14 April 2020).
D luciferin firefly
Manufactured by Biosynth
Sourced in United States, Switzerland
D-luciferin firefly is a laboratory product used for bioluminescence assays. It serves as a substrate for the firefly luciferase enzyme, which catalyzes a light-emitting reaction. This product can be used to quantify the activity of the luciferase enzyme or to detect its presence in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Temozolomide tmz, by MedChemExpress (1 mentions)
Dimethyl sulfoxide (dmso), by Carl Roth (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Carl Roth (1 mentions)
Penicillin streptomycin, by Carl Roth (1 mentions)
Dl threo 1 phenyl 2 palmitoylamino 3 morpholino 1 propanol ppmp, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
Glycerol, by Carl Roth (1 mentions)
Lago in vivo imaging system, by Spectral Instruments Imaging (1 mentions)
Nembutal, by Abbott (1 mentions)
C57bl 6j mice, by Shanghai Laboratory Animal Center (1 mentions)
8 protocols using d luciferin firefly
1
Orthotopic GBM8401-Luc Tumor Model in BALB/c Mice
2
Bioluminescent Tumor Imaging Procedure
3
Comprehensive Immunolabeling Protocol
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The following antibodies were
used: Alexa Fluor 647-labeled anti-6-His epitope tag (Cat. No. 362611)
from BioLegend (San Diego, CA, USA), Anti-Giantin mouse monoclonal
antibody (Cat. No. ab37266) purchased from Abcam (Waltham, Boston,
USA), LAMP1 (D2D11) XP rabbit monoclonal antibody (Cat. No. 9091)
obtained from Cell Signaling Technology (Danvers, Massachusetts, USA),
and Cy3-AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG
(H+L) polyclonal antibody (Cat. No. 711-166-152) and Cy3-AffiniPure
Donkey Anti-Mouse IgG (H+L) polyclonal antibody (Cat. No. 715-165-150)
supplied by Jackson ImmunoResearch (West Grove, Pennsylvania, USA).
The following reagents were obtained from commercial sources: PBS,
FBS, HEPES, NEAA, and 0.05% Trypsin-EDTA (1x) were purchased from
Gibco (Thermo Fisher Scientific Inc., Rockford, IL, USA). DMSO, penicillin/streptomycin,
BSA, DAPI, glycerol, methanol, Triton X-100, and sodium hydrogen carbonate
were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Baden-Württemberg,
Germany).d -Luciferin Firefly was provided by Biosynth (Staad,
Switzerland), and DMEM (with: 1.0 g/L of glucose, stable glutamine,
sodium pyruvate, 3.7 g/L of NaHCO3) was purchased from
PAN Biotech (Aidenbach, Bayern, Germany).dl -Threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol
(PPMP) was obtained from Sigma-Aldrich Chemie GmbH (Saint Louis, MO,
USA).
used: Alexa Fluor 647-labeled anti-6-His epitope tag (Cat. No. 362611)
from BioLegend (San Diego, CA, USA), Anti-Giantin mouse monoclonal
antibody (Cat. No. ab37266) purchased from Abcam (Waltham, Boston,
USA), LAMP1 (D2D11) XP rabbit monoclonal antibody (Cat. No. 9091)
obtained from Cell Signaling Technology (Danvers, Massachusetts, USA),
and Cy3-AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG
(H+L) polyclonal antibody (Cat. No. 711-166-152) and Cy3-AffiniPure
Donkey Anti-Mouse IgG (H+L) polyclonal antibody (Cat. No. 715-165-150)
supplied by Jackson ImmunoResearch (West Grove, Pennsylvania, USA).
The following reagents were obtained from commercial sources: PBS,
FBS, HEPES, NEAA, and 0.05% Trypsin-EDTA (1x) were purchased from
Gibco (Thermo Fisher Scientific Inc., Rockford, IL, USA). DMSO, penicillin/streptomycin,
BSA, DAPI, glycerol, methanol, Triton X-100, and sodium hydrogen carbonate
were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Baden-Württemberg,
Germany).
Switzerland), and DMEM (with: 1.0 g/L of glucose, stable glutamine,
sodium pyruvate, 3.7 g/L of NaHCO3) was purchased from
PAN Biotech (Aidenbach, Bayern, Germany).
(PPMP) was obtained from Sigma-Aldrich Chemie GmbH (Saint Louis, MO,
USA).
4
In Vivo Luciferase Imaging
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Luciferase activity was tracked over time using the Lago in vivo imaging system (Spectral Instruments Imaging) at SCI3. Mice were injected with ~20 mg kg−1d -luciferin Firefly (Biosynth, L-8220) in PBS by retroorbital injection and imaged within 15 min of injection.
5
Bioluminescence Imaging of Luciferase-Expressing Cells
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Bioluminescence imaging was performed with a cryogenically cooled CCD camera (IVIS, Caliper Life Sciences). Acquisition and analysis of images were performed as previously described.45 (link) All animals were imaged 10 min after intraperitoneal (i.p) injection with 100 μL of 40 mg/mL firefly D-luciferin (Biosynth International Inc.). Animals were imaged for 30 s to 5 min, depending on the signal strength. All animals were maintained under isoflurane anesthesia in a 37°C environment.
6
Adenovirus Gene Delivery and Evaluation
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After two days of tail intravenously delivery of Ad-G6p-Luc (108pfu) and Ad-RSV-β-Gal (5 × 107pfu) adenoviruses, mice were orally administered propolis or i.p. injected with APC one dose per day for another 3 days. After 16 h fasting, mice were anesthetized using gaseous isoflurane (Abbott), were i.p. injected with 100 mg/kg sterile firefly D-luciferin (Biosynth AG), and 15 min later, imaged by IVIS-100 Imaging System and analyzed with Living Image Software (Xenogen). Liver lysates were prepared to determine β-Gal activity. Luciferase activity detected for each mouse was normalized to the β-Gal expression in the liver.
7
Bmal1-Luciferase Assays in HEK293T and Primary Hepatocytes
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For cell luciferase activity assays, HEK293T cells were cultured in several 24−well plates before being transfected with Bmal1−luciferase reporter plasmids (400 ng) and RSV−β−gal plasmids (100 ng) using PEI and DMSO or APC (30 μM) overnight and being induced by forskolin (10 nM) for 7 h. Mouse primary hepatocytes were infected with adenoviruses Bmal1−luciferase reporter together with DMSO or APC (30 μM) for 24 h and induced by glucagon (100 nM) for 7 h. Following the induction, cells were collected and detected following a published method [8 (link)].
For liver luciferase activity assays, 5 × 107 pfu Ad−RSV−β−gal and 1 × 108 pfu Ad−Bmal1−lucadenoviruses were injected into mice from the tail vein after the intraperitoneal injection of vehicle or APC (20 mg/kg) on a daily basis for 1 week. After 3–5 days of adenovirus expression, sterile firefly D−luciferin (Biosynth AG, Swiss) at a concentration of 100 mg/kg was injected intraperitoneally into the animals. Then, 3 to 5 min later, an imaging system (IVIS−100) was used to observe the mice. Liver lysates were collected to measure the β−gal activity, which was in turn used to normalize the liver luciferase activity in each well [8 (link)].
For liver luciferase activity assays, 5 × 107 pfu Ad−RSV−β−gal and 1 × 108 pfu Ad−Bmal1−lucadenoviruses were injected into mice from the tail vein after the intraperitoneal injection of vehicle or APC (20 mg/kg) on a daily basis for 1 week. After 3–5 days of adenovirus expression, sterile firefly D−luciferin (Biosynth AG, Swiss) at a concentration of 100 mg/kg was injected intraperitoneally into the animals. Then, 3 to 5 min later, an imaging system (IVIS−100) was used to observe the mice. Liver lysates were collected to measure the β−gal activity, which was in turn used to normalize the liver luciferase activity in each well [8 (link)].
8
Adenovirus Delivery and Bioluminescence Imaging in Mice
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8–10-week-old male C57BL/6J mice were purchased from Shanghai Laboratory Animal Center, CAS and were adapted to colony cages with 12 h light/dark cycle in a temperature-controlled environment for 1 week before study. 1 × 109 plaque forming units (pfu) Ad-Aox-luc; 5 × 107 pfu Ad-RSV-β-gal (Rous sarcoma virus promoter); 3 × 108 pfu Ad-GFP, Ad-SIK2, Ad-p300, Ad-p300 S89A, Ad-p300 LXXAA; 1 × 109 pfu Ad-unspecific RNAi (USi), Ad-SIK2 RNAi (SIK2i), Ad-p300 RNAi (p300i) were delivered by tail-vein injection. For in vivo imaging, mice were fasted for 48 h and refed for 2 h and imaged on day 3–5 after adenovirus delivery. Before imaging, mice were injected intraperitoneally with 50 mg/kg Nembutal (Abbott Laboratories) and 100 mg/kg sterile firefly D-luciferin (Biosynth AG). Mice were imaged on the IVIS 100 Imaging System, and analyzed with Living Image software (Xenogen) as described5 (link). All animal studies were approved by the Animal Experiment Committee of Tongji University and in accordance with the guidelines of school of medicine, Tongji University.
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