Animal experiments were performed as approved by the MSKCC Institutional Animal Care and Use Committee. For orthotopic tumor models, 500 dissociated cells were implanted in 25 μL of Matrigel (Corning Matrigel GFR Membrane Matrix, #356231), injected through 31G syringes into the pancreata of 4-week old female FVB (JAX, FVB/NJ) or athymic nude mice (ENVIGO, Hsd: Athymic Nude-Foxn1nu). Mice were started on 2500 mg/kg doxycycline diet (ENVIGO, TD.07383 2014–2500-B, irradiated) after cell implantation for Tet-On shRNA experiments. Tumor growth was tracked weekly using bioluminescence (Goldbio Firefly D-Luciferin, potassium salt). Tumors were collected from genetic mouse models of PDA (LSL-KrasG12D;Cdkn2afl/fl;Smad4fl/fl and LSL-KrasG12D;Cdkn2afl/fl) for cell lines, organoid lines, and immunohistochemistry. Acute pancreatitis was induced by caerulein injection with 8 hourly injections of 50 μg/kg on 2 consecutive days. For AKT inhibition, mice were dosed by oral gavage with 100 mg/kg MK2206 dissolved in Captisol as a carrier.
Firefly d luciferin
Manufactured by GoldBio
Sourced in United States
Firefly D-luciferin is a chemical compound used as a substrate in bioluminescence assays. It is the primary light-emitting component in the natural bioluminescence of fireflies. When combined with the enzyme luciferase, it produces a luminescent reaction that can be used to measure and detect various biological processes.
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Lab products found in correlation
10 protocols using firefly d luciferin
1
Orthotopic Pancreatic Tumor Models
2
Bioluminescent Imaging of Luciferase Expression
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Imaging for luciferase expression in vivo was performed as previously described [29 (link)]. Briefly, animals were anesthetized with isoflurane (Abbott Laboratories), injected intraperitoneally (I.P.) with firefly D-luciferin (15 mg/mL in PBS; Gold Biotechnology, St. Louis, MO, USA), and imaged 5 min later with a cooled charge-coupled device (CCD) camera (IVIS; PerkinElmer, Waltham, MA, USA). The luciferin dose was 150 mg/kg; the volume varied with the age/size of the animal. Grey-scale photographs were acquired with a 24 cm field of view and a bioluminescence image was obtained using a binning/resolution factor of 4, a 1.2/f stop, and open filter. Regions of interest (ROIs) were defined manually using a standard area for each organ under investigation. Signal intensities were calculated with Living Image software v4.0(Perkin Elmer) and expressed as photons/second/cm2/steradian.
3
Bioluminescence Imaging of Reporter Rodents
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Unless otherwise stated, animals were anesthetized with isoflurane (Abbott Laboratories), injected i.p. with firefly D-luciferin (15 mg/ml in PBS; Gold Biotechnology) and imaged 5 min later with a cooled charge-coupled device (CCD) camera (IVIS; PerkinElmer). Luciferin dose was 150 mg/kg; volume varied with age/size of animal, for example, adult mice were given 300 μl. Grey-scale photographs were acquired with a 24-cm field of view and then a bioluminescence image was obtained using a binning (resolution) factor of 4, a 1.2/f stop and open filter. Regions of interest (ROIs) were defined manually using a standard area for each organ under investigation. Signal intensities were calculated with Living Image software (Perkin Elmer) and expressed as photons per second per cm2 (link) per steradian. Where possible, BLI was carried out in adult reporter rodents on three consecutive days to establish a robust median baseline; subsequent data points were expressed as fold-change over this internal standard for each individual animal.
4
VSV Therapy for CT-26 Tumor Regression
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CT-26 tumors were established by injecting 1 × 106 cells (in 50 μL PBS) subcutaneously into the right hind flank. When tumors reached ∼25 mm2 (∼10 days), mice were treated with labeled (30 μg/mL) or unlabeled VSV (5 × 108 PFU; or PBS control) by tail vein injection. At the indicated time points, mice were injected intraperitoneally (i.p.) with 150 mg/kg firefly D-luciferin (Gold Biotechnology) in PBS, and 10 min later they were imaged using an IVIS system series 100 (Xenogen, Alameda, CA). Photon emission values were calculated using Living Image version (v.)2.5 software (Xenogen). Flank tumor diameters were measured every other day using skin calipers, and mice were euthanized when tumors reached 100 mm2.
5
Adenovirus-Mediated Sirt6 Modulation in Mice
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Adenovirus expressing G6Pase (-231/+57) luciferase (Ad-G6Pase-Luc) was kindly provided by Dr. Um SH (Sungkyunkwan University, Suwon, Korea). Ad-G6Pase-Luc (1 × 109 pfu/kg of body weight) was injected into tail veins of mice. The following day, Sirt6 adenoviruses were intravenously injected into mice, followed by statin treatment for 3 consecutive days. After the last dosing of statin, 6 h-fasted mice were injected with 150 mg/kg of firefly D‐luciferin (GoldBio, St Louis, MO, USA). After 10 min, mice were anesthetized and imaged using the IVIS Luminar XR Imaging System (Caliper Life Sciences, Hopkinton, MA, USA).
6
Bioluminescent Imaging of VSV Infection
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At the indicated time points, mice infected with VSV∆M51-FLUC were injected i.p with 150 mg/kg of firefly D-luciferin (Gold Biotechnology) in PBS and allowed to rest for 10 min. Imaging was conducted using IVIS imaging system series 100 (Xenogen, Alameda, CA, USA) and photon emission values were calculated with Living Image v2.5 software (Xenogen).
7
Murine Models of Lung Cancer
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Five- to six-week-old male C57BL/6 mice and female BALB/c nude mice were purchased from Changzhou Cavens Laboratory Animal Ltd. Mice were raised under specific pathogen-free conditions in the Experimental Animal Center of Tongji Medical College and allowed to adapt to housing in the animal facility for 1 week before the initiation of experiments. Mice were ear-tagged and randomly grouped prior to the experiment. The C57BL/6 mouse lung cancer metastasis model was generated via tail vein injection of LLC-LUC cells. Female BALB/c nude mice were used to establish a subcutaneous tumour model. Animals were photographed using bioluminescence (Goldbio Firefly D-Luciferin, potassium salt, #115144–35-9) under an in vivo optical imaging system (In Vivo FX PRO, Bruker Corporation) at the indicated times to track tumour growth and metastasis.
8
In Vivo Bioluminescence Imaging
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Unless otherwise stated, mice were anaesthetized with isoflurane (Abbott Laboratories), injected i.p. with firefly D-luciferin (15 mg/ml in PBS; Gold Biotechnology) and imaged 5 min later with a cooled charge-coupled device (CCCD) camera (IVIS; PerkinElmer). Luciferin dose was 150 mg/kg; volume varied with age/size of animal, for example, adult mice were given 300μl. Grey-scale photographs were acquired with a 24-cm field of view and then a bioluminescence image was obtained using a binning (resolution) factor of 4, a 1.2/f stop and open filter. Regions of interest (ROIs) were defined manually using a standard area for each organ under investigation. Signal intensities were calculated with Living Image software (Perkin Elmer) and expressed as photons per second per cm2 per steradian. Where possible, BLI was carried out in adult reporter rodents on three consecutive days to establish a robust median baseline; subsequent data points were expressed as fold-change over this internal standard for each individual animal.
9
Luciferase Activity Assay in N. benthamiana
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After agrobacterium-mediated infiltration of N. benthamiana leaves, different light/dark treatments were applied and luciferase activity was detected with a CCD camera by applying firefly D-luciferin (Goldbio). For 24-h light recovery assays, after luminescence imaging, the detached leaves were kept in a tray with water to maintain humidity. After 24 h of light recovery, the luciferase activity was detected with the CCD camera again. Ponceau S Staining (1% (w/v) Ponceau S, 20% acetic acid) was used to quantify relative protein contents.
10
Bioluminescent Imaging of Tumor Progression
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Mice carrying tumors were injected with 150 mg/kg Firefly d-luciferin (Gold Biotechnology, St. Louis, MO) intraperitoneally. Fifteen minutes after luciferin injection, the mice were anesthetized in a plastic chamber filled with 2% isoflurane-air mixture and placed on a warmed stage, after which imaging was performed for 10 seconds to 5 minutes, depending on the tumor model and time point by using the Xenogen IVIS 100 Bioluminescent Imaging System (Perkin Elmer, Boston, MA). Bioluminescent imaging was performed weekly after tumor cell (A549 or H441) injection, depending on the tumor size. The excitation wave length (465 nm) and emission wave length (520 nm) were used to obtain bioluminescent images. The bioluminescent signal was measured in photons per second and quantified using Living Image software (Caliper Life Sciences, Alameda, CA).
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