AGS-GL, BGC-823-GL, KATO III-GL, and MKN-28-GL target cells were incubated with GFP or CAR-MSLN T cells at the indicated ratio in triplicate wells of white 96-well plates. Target cell viability was monitored 18 h later by adding 100 μl/well d -luciferin (potassium salt) (Cayman Chemical, USA) at 150 μg/ml. Background luminescence was negligible (< 1% of the signal from wells containing only target cells). The percent viability (%) was calculated as experimental signal/maximal signal × 100, and the percent lysis was equal to 100% viability.
D luciferin
Manufactured by Cayman Chemical
Sourced in United States
D-luciferin is a bioluminescent compound that emits light when oxidized by the enzyme luciferase. It is a key substrate used in various scientific and research applications, such as in vitro and in vivo imaging of reporter gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Infinite 200 pro, by Tecan (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Ast activity, by Merck Group (2 mentions)
Living image software, by PerkinElmer (2 mentions)
In vivo imaging system spectrum, by PerkinElmer (2 mentions)
Turbofect, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Coelenterazine, by Cayman Chemical (1 mentions)
Coelenterazine h, by Promega (1 mentions)
Nadph, by Cayman Chemical (1 mentions)
Octanoic acid, by Thermo Fisher Scientific (1 mentions)
Ivis lumina xrms in vivo, by PerkinElmer (1 mentions)
Ketamine, by Alfasan (1 mentions)
Isoflurane, by Zoetis (1 mentions)
Lidocaine, by B. Braun (1 mentions)
Pgl4.35 luc2p 9xgal4uas hygro, by Promega (1 mentions)
Caliper ivis lumina 2, by PerkinElmer (1 mentions)
Doxorubicin, by Bio-Techne (1 mentions)
Jnk in 8, by Selleck Chemicals (1 mentions)
37 protocols using d luciferin
1
Cytotoxicity Assay for CAR-T Cell Evaluation
2
In Vivo and Ex Vivo Bioluminescence Imaging
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The IVIS spectrum cooled charge-coupled device optical macroscopic imaging system (Caliper Life Sciences) was used for in vitro and in vivo bioluminescence imaging, as reported by others (48 (link)). In vivo imaging was performed 15 min after i.p. injection of D-luciferin (0.15 mg/g body weight) (Cayman Chemical Company) with the field-of-view set at 13.2 cm, since photon count was most stable during this period. Intensity peaked between 15 and 30 min after i.p. D-luciferin injection. Mice were euthanized 20 min after injection. Each tissue was then isolated, followed by ex vivo bioluminescence imaging. Integration time was fixed at 15 s for each image.
For in vitro imaging, chondrocytes were pellet cultured and treated with D-luciferin (0.5 mM), and images were taken 15 min later. Integration time was fixed at 60 s for each image.
For in vitro imaging, chondrocytes were pellet cultured and treated with D-luciferin (0.5 mM), and images were taken 15 min later. Integration time was fixed at 60 s for each image.
3
Nanoparticle-mediated Therapeutic Delivery and Toxicity Evaluation
4
Luciferase Assay Reagents Protocol
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The chemicals used in the luciferase assay were as follows: FMN-Na (Alfa Aesar, J66949.09), NADPH (Cayman Chemical Company, 9000743), ATP (Larova GmbH, ATP_100ML), D-luciferin (Cayman Chemical Company, 25836), coelenterazine (Cayman Chemical Company, 16123), coelenterazine H (Promega, S2011), frimazine (Aoblous, AOB36539), hispidin (Cayman Chemical Company, 10012605), octanaldehyde (Fisher Scientific, O004425ML), decyl aldehyde (Fisher Scientific, AC154971000), dodecyl aldehyde (fisher scientific), octanoic acid (Fisher Scientific, O002725ML), decanoic acid (Fisher Scientific, AC167271000), dodecanoic acid (Fisher Scientific, S25377), tetradecanoic acid (Fisher Scientific, AAA1206730). D-luciferin, furimazine, and hispidin were dissolved in DMSO as 10 mM stocks. coelenterazine and coelenterazine H were dissolved in ethanol as 10 mM stocks. Long-chain fatty aldehydes and acids were dissolved in ethanol as 500 mM stocks. Dodecyl aldehyde was not soluble in 100% ethanol at the concentration of 500 mM; we used the suspension with vortexing each time.
5
Bioluminescence Imaging of Luciferase Expression
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Luciferase gene expression was quantified using BLI (IVIS Lumina XRMS in vivo imaging system, PerkinElmer, Massachusetts, USA) at various imaging time points indicated in Fig. 1 . Investigators were not blinded to the group allocation during the bioluminescence imaging. Mice were anaesthetised as described above and 50 µl of 15 mg/ml D-luciferin (Cayman Chemicals, US) was delivered to both nostrils as a bolus over 10 to 20 s. Ten minutes later each animal was imaged following a previously described method [10 (link)]. The resultant photon flux was determined in a region of interest created using the auto contour parameter measurement tool with background correction. After the final imaging time point, all mice were humanely killed with sodium pentobarbital (150–300 mg/kg i.p.) while under anaesthetic. Lungs with the trachea attached were immediately excised and re-imaged ex vivo in a small petri dish containing phosphate-buffered saline [10 (link)], to remove the obstructive effect of body tissues and fur on the detectable bioluminescence.
6
Analgesia and Anaesthesia Protocol for In Vivo Imaging
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Pre- and postsurgical analgesia was managed with 67 µg/mL carprofen (Faculty of Veterinary Medicine pharmacy, Utrecht, The Netherlands) per os (p.o.) in drinking water with an additional subcutaneous (s.c.) injection of 5 mg/kg before surgery. Further pain suppression was performed by s.c. injection of 0.5% lidocaine (B. Braun, Melsungen, Germany) during surgery. Anaesthesia was maintained with isoflurane (Zoetis, Capelle aan den IJssel, The Netherlands), mixed with air, 3% induction, 1.8% maintenance. BLI signal of engrafted cells was monitored by intraperitoneal (i.p.) injection of 150 mg/kg D-luciferin (Cayman Chemical, Uden, The Netherlands) in PBS. Euthanasia was performed via i.p. injection of a mix of 7.14 mg/mL ketamine (Alfasan, Woerden, The Netherlands) and 0.714 mg/mL sedazine (AST Farma, Oudewater, The Netherlands) in PBS.
7
Luciferase Assay for RORγ Activity
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The reporter vector pGL4.35[luc2P/9XGAL4UAS/Hygro] was purchased from Promega Cooperation (Madison, WI, USA). The GAL4-DBD RORγ fusion construct was described previously [35 (link)] and was a kind gift from Prof. Patrick Griffin. HEK293 cells were seeded into 96-well white plates at a density of 1 × 104 cells per well. The next day, they were cotransfected with pGL4.35[luc2P/9XGAL4UAS/Hygro], GAL4-DBD RORγ, and pCMV-SEAP (a kind gift from Dr. S. Schlatter, Zurich) vectors with TurboFect (Thermo Fisher Scientific, Waltham, MA, USA). After 24 h, cells were treated with increasing concentrations of the indicated compounds for the next 48 h. Following incubation, the cells were harvested and lysed, and the luciferase activity was determined using an Infinite® 200 PRO (Tecan Group) with D-Luciferin (luciferase substrate) (Cayman Chemical Company). Alkaline phosphatase activity was determined spectrophotometrically at 405 nm in culture medium as a control of transfection efficiency.
8
In Vivo Tumor Growth Imaging
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To accurately track changes in in vivo tumor growth with the treatments, we performed bioluminescence analysis using the Caliper IVIS Lumina II imaging system at CMCA, UWA. The analyses were conducted every 2 days after the generation of tumors. Mice were injected intraperitoneally with 200 µL of D-Luciferin (Cayman Chemical) at the final concentration of 150 mg/kg dissolved in PBS prior to being anesthetized at 4% isoflurane. Once anesthetized, mice were placed inside the prewarmed chamber of the bioluminescence imager and imaged 7–12 min after injection, under 2% isoflurane, until bioluminescence signal intensity had reached a steady state.
9
Evaluating Tumor Immunotherapies in Mice
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For subcutaneous tumors, C57BL/6 mice were vaccinated with OVA (10 μg), OVA (10 μg) + MnJ (20 μg), or OVA (10 μg) + aluminum (1320 μg) on days 0, 7, and 14. On day 21, vaccinated animals were inoculated subcutaneously in the right hind flank with 3 × 105 B16-F10-OVA cells. Tumor sizes were measured every 2 days with electronic calipers and calculated by length (mm) × width (mm) × width (mm)/2. Mice with tumors larger than 20 mm on the longest axis were euthanized. IVIS images were captured with a Caliper IVIS Lumina II (Caliper Life Sciences) instrument following light anesthesia with pentobarbital sodium and intraperitoneal injection of D-luciferin (Cayman Chemical, 14681) (0.5 mg/g per mouse). Images were quantified using Living Image 4.0.
For metastatic tumors, C57BL/6 mice were vaccinated with OVA (10 μg) or OVA (10 μg) + MnJ (20 μg) on days 0, 7, and 14. On day 21, vaccinated animals were inoculated intravenously with 3 × 105 B16-F10-OVA-GFP cells. Mice were euthanized 20 days after inoculation. Images of lungs and HE-stained lung sections were recorded.
For metastatic tumors, C57BL/6 mice were vaccinated with OVA (10 μg) or OVA (10 μg) + MnJ (20 μg) on days 0, 7, and 14. On day 21, vaccinated animals were inoculated intravenously with 3 × 105 B16-F10-OVA-GFP cells. Mice were euthanized 20 days after inoculation. Images of lungs and HE-stained lung sections were recorded.
10
GPC3-CAR T Cell Cytotoxicity Assay
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The target cells HepG2-GL, Huh-7-GL, and A549-GL were incubated with Control-CAR T or GPC3-CAR T cells at the indicated ratios in triplicate wells in U-bottomed, 96-well plates. Target cell viability was monitored 24 h later by adding 100 µL/well substrate d -Luciferin (potassium salt; Cayman Chemical, USA) resolved at 150 µg/mL. The background luminescence was negligible (<1% the signal from the wells with only target cells). The viability percentage (%) was, therefore, equal to the experimental signal/maximal signal, and the killing percentage was equal to 100 − viability percentage.
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