Experiments were performed using the LANCE Ultra cAMP kit (Perkin Elmer) with cells in suspension according to the manufacturer’s instructions with minor modifications. Briefly, cells were harvested using versene (Gibco, Thermo Scientific), spun at 100×g for 5 minutes and resuspended in assay buffer (HBSS, 5 mM HEPES, 0.5 mM IBMX (Sigma-Aldrich), 0.1% BSA, pH 7.4) at 8,000 cells/well for hNPSR-107N, 4,000 cells/well for hNPSR-107I, and 20,000 cells/well for mNPSR. Serial dilutions of test compounds and NPS were prepared at 2× the desired final concentration in assay buffer. Cells were transferred to 96-well ½ area white polystyrene plates, and NPS or test compounds were added at the indicated concentrations. Plates were incubated for 30 minutes (hNPSR-107N or hNPSR-107I) or 1 hour (mNPSR) at room temperature. After addition of Eu-tracer cAMP and ULight-anti-cAMP, plates were incubated for 1 hour at room temperature in the dark. Data was collected on a FlexStation III instrument (excitation at 340 nm, emission at 615 and 665 nm, Molecular Devices) or a CLARIOstar (excitation at 340 nm, emission at 620 and 665 nm, BMG LABTECH). TR-FRET data in relative fluorescent units (RFU) was converted to fmol cAMP through interpolation using a cAMP standard curve and data were fit using a three-parameter non-linear regression to generate EC50 values (GraphPad Prism, 6.0).
Flexstation 3 instrument
Manufactured by Molecular Devices
The FlexStation III instrument is a multi-mode microplate reader designed for versatile applications in life science research. It provides automated, high-throughput detection of fluorescence, luminescence, and absorbance within microplates. The instrument is capable of performing rapid kinetic measurements, endpoint assays, and spectral scanning to support a wide range of biological and biochemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Nano glo luciferase assay kit, by Promega (2 mentions)
Fugene hd transfection reagent, by Promega (2 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Lance ultra camp kit, by PerkinElmer (1 mentions)
Versene, by Thermo Fisher Scientific (1 mentions)
Prism 6, by GraphPad (1 mentions)
Clariostar, by BMG Labtech (1 mentions)
Calcium 5 assay kit, by Molecular Devices (1 mentions)
Prism 9, by GraphPad (1 mentions)
4 protocols using flexstation 3 instrument
1
cAMP Quantification in NPSR Cells
2
Calcium Flux Assay for H3R Mutants
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Calcium flux was performed as described in our previous studies. Briefly, CHO cells were co-transfected with wild-type or mutant H3R and Gqi5 using Lipofectamine 2000 according to the manufacturer’s manual. Transfected cells were seeded into a 96-well flat clear bottom black plate with a density of 25,000 cells per well and cultured overnight. Subsequently, cells were loaded with calcium dye solution from Calcium 5 assay kit (Molecular Devices) in Hanks’ balanced salt solution (20 mM HEPES, 2.5 mM probenecid in HBSS), and incubated at 37 °C for 45 min. Various concentrations of compounds were dispensed into the wells via a Flexstation III instrument (Molecular Devices). The intracellular calcium flux was detected immediately using the Flexstation III instrument (excitation at 485 nm, emission at 525 nm). Data were representative of three independent experiments and analyzed using GraphPad Prism 9.3.1.
3
SARS-CoV-2 Spike Pseudovirus Assay
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Pseudoviruses were packaged in HEK293T cells by cotransfecting the retroviral vector (retrovector) pMIG (kindly provided by Jianhua Li, Fudan University) in which the target gene was replaced with the nanoluciferase gene, a plasmid expressing murine leukemia virus (MLV) Gag-Pol, and pcDNA3.1 expressing SARS-CoV-2 spike genes or vesicular stomatitis virus G (VSV-G) (pMD2.G; Addgene plasmid 12259) using Fugene HD transfection reagent (Promega). At 48 h posttransfection, the supernatant was harvested, clarified by spinning at 3,500 rpm for 15 min, aliquoted, and stored at −80°C. Virus entry was assessed by the transduction of pseudoviruses in 96-well plates. After 16 h, the luciferase activity was determined using a Nano-Glo luciferase assay kit (catalog no. N1110; Promega) according to the manufacturer’s instructions. The same volume of reagent was added to each well, and the mixture was shaken for 2 min. After incubation at room temperature for 10 min, luminescence was recorded by using a FlexStation 3 instrument (Molecular Devices), with an integration time of 1 s per well.
4
SARS-CoV-2 Spike Pseudovirus Assay
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Pseudoviruses were packaged in HEK293T cells by cotransfecting the retroviral vector (retrovector) pMIG (kindly provided by Jianhua Li, Fudan University) in which the target gene was replaced with the nanoluciferase gene, a plasmid expressing murine leukemia virus (MLV) Gag-Pol, and pcDNA3.1 expressing SARS-CoV-2 spike genes or vesicular stomatitis virus G (VSV-G) (pMD2.G; Addgene plasmid 12259) using Fugene HD transfection reagent (Promega). At 48 h posttransfection, the supernatant was harvested, clarified by spinning at 3,500 rpm for 15 min, aliquoted, and stored at −80°C. Virus entry was assessed by the transduction of pseudoviruses in 96-well plates. After 16 h, the luciferase activity was determined using a Nano-Glo luciferase assay kit (catalog no. N1110; Promega) according to the manufacturer’s instructions. The same volume of reagent was added to each well, and the mixture was shaken for 2 min. After incubation at room temperature for 10 min, luminescence was recorded by using a FlexStation 3 instrument (Molecular Devices), with an integration time of 1 s per well.
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