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10 protocols using ip one tb htrf kit

1

Measuring Inositol 1-Phosphate Signaling

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Inositol 1-phosphate (IP-1), a metabolite of inositol trisphosphate, which is downstream of signaling by Gαq or Gβγ subunits, was detected using the IP-One Tb HTRF kit (Cisbio Bioassays, Bedford, MA), as described elsewhere earlier [20 (link)]. Cells were grown in 96-well plates overnight before the pretreatment with UBO-QIC (100 nM) for 30 min before the addition of agonists followed by additional 30 min incubation. Assay plates were read on a Mithras LB940 reader (Berthold Technologies, Oak Ridge, TN) or a PerkinElmer (Waltham, MA) EnSpire plate reader using a time-resolved fluorescence ratio (665/620 nm).
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2

Measuring IP-1 Levels in U2Os Cells

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Inositol 1-phosphate (IP-1) was measured using the IP-One Tb HTRF kit (Cisbio Bioassays, Bedford, MA) as described previously60 (link),61 (link). Briefly, after overnight growth, U2Os cells expressing the P2Y1 receptor were first treated with an antagonist for 20 min before the treatment with agonist and incubated for another 60 min. IP-1 detection reagents were added as instructed by the manual from the manufacturer. The assay plates were read on a Mithras LB940 reader (Berthold Technologies, Oak Ridge, TN) using a time-resolved fluorescence ratio (665/620 nm).
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3

Measuring GPCR-Mediated IP1 Accumulation

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Total inositol 1 phosphate (IP1) level was measured 48 h after transfection in adherent cells plated in a 384-well white opaque plate at a density of 14,000 cells/well. CHO-M1 cells (CHO cells stably expressing the muscarinic receptor) were transfected with MRAP2 alone, GHSR1a and empty vector, or GHSR1a and MRAP2 at a 1:5 ratio and plated in a 384-well plate the next day. IP1 accumulation was measured using the IP-One Tb HTRF kit (Cisbio Bioassays) according to the manufacturer’s instructions. Signals at 665 and 620 nm were detected using Spectramax i3 (Molecular Devices) equipped with a Cisbio HTRF module. Results were normalized to the signal obtained in response to 10 µM Carbachol for each condition.
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4

Purinergic Receptor Assays and Ligands

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[[(1R,2R,3S,4R,5S)-4-[6-Amino-2-(methylthio)-9H-purin-9-yl]-2,3-dihydroxybicyclo[3.1.0]hex-1-yl]methyl] diphosphoric acid monoester trisodium salt (MRS2365) was from Tocris (St. Louis, MO). N6-Cyclopentyladenosine (CPA), carbachol, PTX and 2-methylthioadenosine 5′-diphosphate trisodium salt (2MeSADP), adenosine-5′-N-ethyluronamide (NECA) were from Sigma (St. Louis, MO). UBO-QIC was purchased from University of Bonn (Germany). IP-One Tb HTRF kit was from Cisbio Bioassays (Bedford, MA). AlphaScreen cAMP kit, SureFire p-ERK1/2 (Thr202/Tyr204) Assay Kit and AlphaScreen SureFire p-Akt 1/2/3 (p-Ser473) Assay Kit were purchased from PerkinElmer (Waltham, MA). HEK293 and DDT1-MF2 were from ATCC (Mannasas, VA); CHO cell lines stably expressing the human A1AR, A2AAR, and A2BAR, and human M3 and M2 muscarinic acetylcholine receptors were made at the Laboratory of Bioorganic Chemistry, NIDDK (Bethesda, MD). 1321N1 astrocytoma cells expressing either the human P2Y1R or P2Y12R were from T. K. Harden (University of North Carolina, Chapel Hill, NC); all other reagents were from standard commercial sources and of analytical grade.
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5

MRAP2 Modulation of GPCR Signaling

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Inositol phosphate assay was performed using the IP-One-Tb HTRF kit (Cisbio Bioassays, France) following the manufacturer’s instructions. CHO-M1 cells were transfected with PKR1 or PKR2 plus either empty vector or MRAP2 at a ratio of 1:10 (receptor to MRAP2). The day before the experiment, the transfected cells were plated in a white 384-well plate at a density of 20,000 cells/well. The next day the cells were treated with different concentrations of PK1, PK2 or 1 μM carbachol in stimulation buffer containing lithium for 1 hr at 37°C, and then incubated with IP1-d2 conjugate and Anti-IP1 cryptate Tb in lysis buffer for 1 hr at room temperature. HTRF signal was read at 615 nm and 665 nm using a Spectramax I3 plate reader equipped with the HTRF Cisbio cassette. For each condition, signal obtained with 1 μM carbachol, an agonist of the M1 muscarinic receptor, was used for normalization. Results were then normalized to the highest signal from the control (receptor + empty vector).
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6

Evaluating mGluR Receptor Activation in HEK293 Cells

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HEK293 cells were transfected and seeded and to the point of assay initiation treated as described above except that cells were seeded in clear poly-L-lysine coated 96-well plates. Following the wash, 50 μL HBSS buffer was added to each well. MPEP (Sigma M5435) or buffer were preincubated by addition of MPEP or buffer in 50 µL HBSS buffer for 15 min at 37°C before adding the orthosteric and/or allosteric activating ligands (or vehicle condition) also diluted in 50 µL HBSS buffer now supplemented with LiCl to a final concentration of 20 mM. After incubation for 30 min at 37°C the reaction was stopped by addition of 40 µL lysis buffer from the CisBio IP-One Tb HTRF Kit (CisBio 62IPAPEC). The accumulated IP1 levels were then quantified according to the manufactures instructions and quantified using an Envision plate reader (Perkin Elmer). Data were calculated as the amount of IP1 formed per well or normalized to the basal IP1 level and fitted by non-linear regression using GraphPad Prism.
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7

Evaluating mGluR Receptor Activation in HEK293 Cells

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HEK293 cells were transfected and seeded and to the point of assay initiation treated as described above except that cells were seeded in clear poly-L-lysine coated 96-well plates. Following the wash, 50 μL HBSS buffer was added to each well. MPEP (Sigma M5435) or buffer were preincubated by addition of MPEP or buffer in 50 µL HBSS buffer for 15 min at 37°C before adding the orthosteric and/or allosteric activating ligands (or vehicle condition) also diluted in 50 µL HBSS buffer now supplemented with LiCl to a final concentration of 20 mM. After incubation for 30 min at 37°C the reaction was stopped by addition of 40 µL lysis buffer from the CisBio IP-One Tb HTRF Kit (CisBio 62IPAPEC). The accumulated IP1 levels were then quantified according to the manufactures instructions and quantified using an Envision plate reader (Perkin Elmer). Data were calculated as the amount of IP1 formed per well or normalized to the basal IP1 level and fitted by non-linear regression using GraphPad Prism.
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8

HTRF Assay for Inositol Phosphate

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Inositol phosphate (IP) accumulation was measured using a homogenous time-resolved FRET (HTRF) assay (HTRF IP-One Tb kit, Cisbio Bioassays, Codolet, France). HEK293T cells were transiently cotransfected with FLAG-hGPR35-eYFP and the G-protein chimaera Gαq/135 (a form of Gαq in which the C-terminal 5 amino acids were replaced with the corresponding pentapeptide from Gα13) using PEI. After 16-24 h of incubation at 37°C in a 5% CO2 humidified atmosphere, the cells were resuspended in IP-One stimulation buffer (10 mM HEPES, 1 mM CaCl2, 0.5 mM MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM glucose and 50 mM LiCl, pH7.4) and seeded at 10,000 cells/well in white, solid-bottom, 384-well plates. Ligands were diluted in IP-One stimulation buffer according to the manufacturer's instructions. Antagonist compounds were preincubated with cells for 15 min at 37°C prior to the addition of the agonist. Cells were incubated with ligand(s) for 2 h at 37°C, before the addition of d2-conjugated IP monophosphate (IP1; 3 μl/well) and anti-IP1 Lumi4™-Tb cryptate (3 μl/well) diluted in lysis buffer. After incubation at room temperature for 1 h, HTRF was measured using a PHERAstar FS plate reader (BMG-Labtech). IP1 accumulation was measured by the fluorescence ratio of 665 nm/620 nm.
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9

HTRF Assay for IP1 Accumulation

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HEK 293 T cells were seeded into T75 flasks at 90,000 cells/cm2 and incubated at 37 °C and 5% CO2 overnight. The following day cells were transiently transfected for 24 hours. IP1 accumulation was measured using Homogeneous Time-Resolved Fluorescence (HTRF) IP-ONE Tb Kit (Cisbio, Codolet, France) as per manufacturer’s instruction. The assay was perfomed using 10,000 cells/well into white, low volume 384-well plates (Greiner Bio-One) The HTRF signal was read on an Envision imager (Perkin-Elmer) after a 1 hour room temperature incubation.
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10

Evaluating GLUTag Cell Metabolism

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GLUTag cells were seeded for 2 d and then treated with 50 μM AzA or 10 μM forskolin for 15 min and 30 min for cAMP measurement and PKA activity assay, respectively. cAMP concentrations were assayed using a fluorimetric ELISA kit (AAT Bioquest, Sunnyvale, CA, USA). PKA kinase activity was measured using a commercial kit from Enzo Life Science (Farmingdale, NY, USA). Intracellular calcium was quantified with Fluo-4 Direct Ca2+ reagent (Invitrogen, USA), and inositol phosphates were measured with a HTRF IP-one Tb kit (Cisbio, Bedford, MA, USA), according to the manufacturer’s protocol. Cell proliferation was analyzed using the CellTiter-Glo 2.0 kit (Promega, Madison, WI). Mitotracker Green probe (Molecular Probes) was used to measure the mitochondrial biogenesis in GLUTag cells following the manufacturer’s instructions. Briefly, GLUTag cells were incubated with Green probes (200 nM) for 30 min at 37°C after washing with PBS (pH 7.4). Subsequently, the green fluorescence intensity was measured using SpectraMAX (Molecular Devices Co.) at the wavelength of 490 nm (excitation) and 516 nm (emission), respectively. Apoptosis assay was performed in GLUTag cells at 24 h post treatment using the Caspase-Glo 3/7 assay kit (Promega) according to the manufacturer’s instructions.
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