The inositol phosphate IP1 accumulation assay was applied to evaluate the Gq signals of MTLR. IP1 production was measured using the IP-One HTRF kit (Cisbio). Briefly, AD-293 cells (Agilent) were grown to a density of 400,000 to 500,000 cells/ml and then infected with separate plasmids at a suitable concentration. The culture was collected by centrifugation 24 hours after incubation at 37°C in 5% CO2 with a stimulation buffer. The cell suspension was then dispensed in a white 384-well plate at a volume of 7 μl per well before adding 7 μl of ligands. The mixture was incubated for 1 hour at 37°C. IP1-d2 and anti-IP1 cryptate dissolved in lysis buffer (3 μl each) were subsequently added and incubated for 15 to 30 min at room temperature before measurement. Intracellular IP1 measurement was carried out with the IP-One HTRF kit and EnVision multi-plate reader (PerkinElmer) according to the manufacturer’s instructions. Data were normalized to the baseline response of the ligand.
Ip one htrf kit
Manufactured by PerkinElmer
Sourced in France
The IP-One HTRF kit is a laboratory equipment product from PerkinElmer designed to measure inositol phosphate levels in cells. It utilizes Homogeneous Time-Resolved Fluorescence (HTRF) technology to quantify the formation of inositol-1-phosphate, a messenger molecule involved in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Envision multiplate reader, by PerkinElmer (7 mentions)
Ad293 cells, by Agilent Technologies (6 mentions)
Prism 8, by GraphPad (2 mentions)
Quisqualate, by Bio-Techne (1 mentions)
Prism software, by GraphPad (1 mentions)
Stimulation buffer, by PerkinElmer (1 mentions)
Dmem glutamax 1, by Thermo Fisher Scientific (1 mentions)
White 384 well plate, by Greiner (1 mentions)
Spark fluorescence plate reader, by Tecan (1 mentions)
Phenylephrine, by Bio-Techne (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
14 protocols using ip one htrf kit
1
Measuring G protein-coupled receptor activation via IP1 assay
2
Measuring Intracellular IP-One Levels
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IP-One production was measured using the IP-One HTRF kit (Cisbio)62 . Briefly, AD293 cells (Agilent) were grown to a density of 400,000–500,000 cells per mL and then infected with separate plasmids at a suitable concentration. The culture was collected by centrifugation 24 h after incubation at 37 °C in 5% CO2 with a Stimulation Buffer. The cell suspension was then dispensed in a white 384-well plate at a volume of 7 μl per well before adding 7 μl of ligands. The mixture was incubated for 1 h at 37 °C. IP-One-d2 and anti-IP-One Cryptate dissolved in Lysis Buffer (3 μl each) were subsequently added and incubated for 15-30 min at room temperature before measurement. Intracellular IP-One measurement was carried with the IP-One HTRF kit and EnVision multi-plate reader (PerkinElmer) according to the manufacturer’s instructions. Data were normalized to the baseline response of the ligand.
3
Measurement of IP-One Production in AD293 Cells
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IP-One production was measured using the IP-One HTRF kit (Cisbio)50 . Briefly, AD293 cells (Agilent) were grown to a density of 400,000–500,000 cells per mL and then infected with separate plasmids at a suitable concentration. The culture was collected by centrifugation 24 h after incubation at 37 °C in 5% CO2 with a Stimulation Buffer. The cell suspension was then dispensed in a white 384-well plate at a volume of 7 μl per well before adding 7 μl of ligands. The mixture was incubated for 1 h at 37 °C. IP-One-d2 and anti-IP-One Cryptate dissolved in Lysis Buffer (3 μl each) were subsequently added and incubated for 15-30 min at room temperature before measurement. Intracellular IP-One measurement was carried out with the IP-One HTRF kit and EnVision multi-plate reader (PerkinElmer) according to the manufacturer’s instructions. Data were normalized to the baseline response of the ligand. pEC50Emin, and Emax for each curve were calculated by GraphPad Prism 8.0. ∆pEC50 equals pEC50 of agonists to specific Mutant minus pEC50 of agonists to WT. Data are presented as mean values ± SEM; n = 3 independent samples; n.s. no significant; *p < 0.05; **p < 0.01; ***p < 0.001.
4
HTRF-Based IP1 Accumulation Assay
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IP1 accumulation was measured using the IP-One HTRF kit (PerkinElmer, CisBio Bioassays). Transfected HEK293 cells were seeded in a 96-well plate, and 24 h after transfection, cells were treated with baclofen diluted in stimulation buffer in a Cisbio kit for 30 min at 37 °C. Then, cryptate-labelled anti-IP1 monoclonal antibody and d2-labelled IP1 in lysis buffer were added to the wells. After 1 h of incubation at room temperature, the plates were read in PHERAstar FS with excitation at 337 nm and emission at 620 and 665 nm. The accumulation of IP1 was calculated according to a standard dose–response curve.
5
GPCR Signaling Pathway Quantification
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The day of the experiment, media were replaced by stimulation buffer with LiCl to prevent IP1 degradation (NaCl, 146 mM, KCl, 4.2 mM, MgCl2, 0.5 mM, CaCl2, 1 mM, HEPES, 10 mM, glucose, 5.5 mM, LiCl, 50 mM, pH7.4). Cells were stimulated during 2 h at 37°C with different concentrations of BW (10−11 to 10−6 M in stimulation buffer). Stimulation solution was replaced by a lysis buffer (IP one HTRF Kit, Cisbio, France) during 1 h. Lysates were distributed to 384-well plates, and IP was labeled using HTRF reagents. The assay is based on a competitive format involving a specific antibody labeled with terbium cryptate (donor) and IP coupled to d2 (acceptor). After a 1-h incubation with HTRF reagent, the plate was read using Mithras LB940 plate reader according to the manufacturer’s instructions. Modelization and EC50 calculation were done using GraphPad Prism 7 software. At least three independent experiments were performed in duplicate.
6
MCHR2-Mediated Gαq Signaling Quantification
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IP1 accumulation assay was applied for the detection of the downstream Gαq signal of MCHR2 using the IP-One HTRF kit (Cisbio). AD293 cells were cultured in DMEM/high glucose supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin–streptomycin at 37 °C in 5% CO2. After transfection for 24 h, cells were harvested, resuspended with 1× stimulation buffer to a density of 8 × 105 cells/mL, and then seeded to 384-well plates for 7 μL/well. After dispensing 7 μL different concentrations of ligand diluted with 1× stimulation buffer, the mixture was incubated at 37 °C for 1 h. IP1-d2 and anti-IP1 cryptate were dissolved in 1× lysis buffer and sequentially added to the plates for 3 μL/well. Before measurement, the samples were incubated at RT for 30 min and measured with EnVision multi-plate reader (PerkinElmer).
7
Measurement of Inositol Monophosphate Accumulation
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The extent of inositol monophosphate (IP1) accumulation was measured using a IPOne HTRF kit (Cisbio Bioassays, cat. no. 62IPAPEJ) according to the recommendations of the manufacturer. Briefly, cells were seeded and transiently transfected with CB1R, or the variant, together with chimeric G protein Gqi9 (1:1 ratio). Gqi9 allows Gi/o‐coupled GPCRs to couple to Gq to produce IP1(Brule et al., 2014 (link)). Twenty‐four hours after the transfection, cells were incubated in the presence of receptor agonist for 20 min at 37°C, and then cryptate‐labeled anti‐IP1 and D2‐labeled IP1 antibodies were added for 1 h at the 21°C. Native IP1 produced by cells compete with d2‐labeled IP1 (acceptor of energy) for binding of anti‐IP1‐Cryptate (donor of energy). The fluorescence was detected at 665 and 620 nm using a PHERAstar plate reader (BMG Labtechnologies, Germany). The HTRF signal was calculated as the ratio of 665/620 nm emission multiplied by 10,000. The specific measured HTRF signal (energy transfer) is inversely proportional to the concentration of IP1 in the cells. The data were normalized against the minimal and maximal IP1 accumulation in cells driven by specific CB1R variant.
8
Measuring IP Accumulation in HEK293 Cells
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IP accumulation in HEK293 cells was measured using the IP-One HTRF kit (Cisbio Bioassays) according to the manufacturer’s recommendations.
9
Monitoring Glutamate Receptor Activity
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HEK293 cells were transiently transfected with mGlu5 receptors by electroporation. Receptors were cotransfected with EAAC1, a glutamate transporter, to avoid the influence of extracellular glutamate. The receptor activity was monitored through measurements of inositol monophosphate (IP1) production. IP1 production was determined using the IP-One HTRF kit (CisBio Bioassays) according to the manufacturer’s recommendations [48 (link)]. All points were performed in triplicate. Mutant receptors were obtained using the Quick-Change strategy (Stratagene, La Jolla, CA, USA) and all mutations were verified by sequencing. Experimental data were fitted using the Hill equation with Prism software (GraphPad, La Jolla, CA, USA). Quisqualate and MPEP were purchased from Tocris Bioscience (Bristol, UK). Alloswitch-1 was synthesized in the laboratory of A. Llebaria following described literature procedures [29 (link)]. All solutions were prepared just before experiments.
10
Measurement of mGlu5 Receptor IP Accumulation
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The mGlu5-mediated inositol phosphate (IP) accumulation was determined using the FRET-based assay IP-One HTRF kit (Cisbio Bioassays) according to the manufacturer’s instructions (Trinquet et al., 2006 (link)). Glutamate levels were maintained at minimal concentrations by co-expressing the excitatory amino acid transporter one (EAAC1). Importantly, all the mGlu5 receptor contained a haemagglutinin (HA) epitope tag in their N terminus to allow cell surface expression by ELISA for subsequent normalization. After transfection cells were seeded in black clear-bottom 96-well plates at a concentration of 1.5 × 105 cells/well. The medium was replaced by glutamate-free DMEM GlutaMAX-I (Invitrogen) after 6 hr. Next day cells were challenged with increasing concentrations of the test compounds in the presence of quisqualate EC80 (1 µM) for 30 min, at 37°C and 5% CO2 before we determined IP accumulation (Trinquet et al., 2006 (link)). When necessary the compounds were irradiated at 405 nm for 5 min before being added to the cells. Finally, the cells were washed once with stimulation buffer (Cisbio Bioassays) to remove any interfering chromophore and then the cells were lysed for IP determination. The IP associated fluorescence was determined in a RUBYstar multimode microplate reader (BMG Labtech).
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