CHO cells transfected with NK2 and NK1 receptors showed negligible cAMP stimulation when treated with NKA or SP (data not shown). The luciferase reporter gene assay, which uses response-element-regulated expression of luciferase, has been widely applied to high-throughput functional assays for many targets because its high sensitivity and reliability [8 (link)]. Therefore, to increase the sensitivity of detection for cAMP signaling, CHO cells expressing NK2 or NK1 receptors were transiently transfected with a CRE-LUC reporter construct using Lipofectamine 2000 (Life Technologies, Carlsbad, USA). At 24 h post-transfection, cells were transferred to a 96-well culture plate and stimulated with culture medium containing 7 concentrations of GR64349, NKA, or substance P (0.19 pM - 1 μM). Luciferase expression was measured after 5 h of incubation by adding Firefly Luciferase reagent, followed by Renilla Luciferase reagent (BPS Bioscience, San Diego, USA). Luminescence was measured by using an Envision Multilabel flash lamp reader after addition of the first and second reagents. Each concentration was tested in duplicate in 4 experiments.
Renilla luciferase reagent
Manufactured by BPS Biosciences
Sourced in United States
Renilla Luciferase reagent is a bioluminescent substrate used to measure the activity of the Renilla luciferase reporter enzyme. It is a core component in various cell-based assays and reporter gene studies.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Firefly luciferase reagent, by BPS Biosciences (2 mentions)
F 12k medium, by Thermo Fisher Scientific (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Geneticin g418, by Thermo Fisher Scientific (1 mentions)
Sigmafast protease inhibitor tablet, by Merck Group (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Hepes, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
Bacitracin, by Merck Group (1 mentions)
Substance p, by Merck Group (1 mentions)
3 isobutyl 1 methylxantine, by Merck Group (1 mentions)
Microscint 20, by PerkinElmer (1 mentions)
125i nka, by PerkinElmer (1 mentions)
2 protocols using renilla luciferase reagent
1
Functional Screening of NK Receptors
2
Synthesis and Characterization of NKA Analogs
Check if the same lab product or an alternative is used in the 5 most similar protocols
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NKA and the peptide analogs of NKA listed in Table 1 were synthesized by Genscript (Piscataway, USA) to a purity ≤95%. NKA and substance P were purchased from Sigma Aldrich, (St. Louis, USA); septide was purchased from Bachem (Bubendorf, Switzerland, EU). [3H]-Septide was custom synthesized from Quotient Bioresearch (Cardiff, UK). [125I]-NKA, and Microscint 20, were obtained from Perkin Elmer (Boston, USA). F12K medium, Geneticin (G418) and Lipofectamine 2000 were obtained from LifeTechnologies (Carlsbad, USA). Phosphate buffered saline (PBS) was from Lonza (Walkersville, MD, USA) and EDTA from Life Technologies (New York, USA). FuGENE HD Transfection Reagent was obtained from Promega (Madison, USA). The following reagents were obtained from Sigma Aldrich (St. Louis, USA): DMSO, Pluronic F-127, HEPES, bacitracin, SIGMAFAST Protease Inhibitor Tablets, bovine serum albumin (BSA), polyethileneimine (PEI), NaCl, 3-isobutyl-1-methylxantine (IBMX), and probenecid. The CRE/CREB Reporter Assay Kit, Firefly Luciferase reagent, and Renilla Luciferase reagent were purchased from BPS Bioscience (San Diego, USA).
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