The IP-One assay kit (Cisbio, France) was used for the direct quantitative measurement of myoinositol 1-phosphate (IP1) in FlpIn CHO cells stably expressing hM1 and hM3 mAChRs. This is a competitive immunoassay that measures the homogeneous time-resolved fluorescence signal transferred between a cryptate-labeled IP1-specific monoclonal antibody and d2-labeled IP1. The fluorescence signal measured is inversely proportional to the concentration of native IP1. Briefly, cells were seeded into 96-well plates at 20000 cells per well and allowed to grow overnight at 37 °C, 5% CO2. The following day, cells were washed once with PBS then incubated with stimulation buffer (HEPES 10 mM, CaCl2 1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM, LiCl 50 mM, pH 7.4) for 60 min at 37 °C, 5% CO2. Following this incubation, ligands were added at 10× their final concentrations (ACh or test compounds) and incubated for a further 40 min prior to terminating the ligand-mediated stimulation by removing the buffer and adding 25 μL of lysis buffer. Finally, 14 μL of lysate was transferred into 384-well Optiplate, followed by the addition of 3 μL of IP1-d2, then 3 μL of Ab-Cryp, and incubated for 60 min at room temperature. Time resolved fluorescence resonance energy transfer (HTRF) was determined using the Envision plate reader (PerkinElmer).
Ip one assay kit
Manufactured by PerkinElmer
Sourced in France
The IP-One assay kit is a laboratory equipment product from PerkinElmer. It is designed to measure inositol monophosphate (IP1), which is a downstream metabolite of the inositol phosphate signaling pathway. The kit provides a quantitative analysis of IP1 levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Envision plate reader, by PerkinElmer (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Flexstation 3 plate reader, by Molecular Devices (1 mentions)
Spark multimode plate reader, by Tecan (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by GE Healthcare (1 mentions)
Atosiban, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
7 protocols using ip one assay kit
1
IP1 Quantification in Transfected Cells
2
Quantitative IP1 Receptor Assay Protocol
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Quantitative measurements of receptor-mediated inositol 1-phosphate (IP1) were performed by competitive immunoassay utilising the IP-One assay kit (CisBio, France). HEK293 cells were prepared as described above and then assayed as per the manufacturer’s protocol. Briefly, at the time of assay, the culture media was removed from cells, and then replaced with IP-One stimulation buffer (HEPES 10 mM, CaCl2 1 mM, MgCl2 0.5 mM, KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM, LiCl 50 mM, pH 7.4). Cells were allowed to equilibrate in the stimulation buffer at 37 °C for 15 min, followed by addition of peptide ligands (to final concentrations of 0.1 pM – 10 µM) for subsequent stimulation for 1 h at 37 °C. The stimulation was terminated by lysis and the simultaneous addition of homogenous time-resolved fluorescence resonance energy transfer reagents. The lysates were incubated for a minimum of 1 h at room temperature. Fluorescence emission measurements at 620 nm and 665 nm were performed using a FlexStation3 plate reader (Molecular Devices, Sunnyvale, CA) or Spark Multimode plate reader (Tecan, Männedorf, Switzerland) at an excitation wavelength of 340 nm. Results were analysed as a ratio of fluorescence intensities of 665 nm to 620 nm.
3
HEK293T Cells Transfection and IP-one Assay
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HEK293T cells were cultured in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO2. The cells were transiently transfected with wild-type or mutant NK3R using Lipofectamine 3000 (Thermo Fisher Scientific). Twenty-four hours post transfection, IP-one experiments were performed using the IP-one assay kit (Cisbio, 62IPAPEC) according to the kit instructions. In brief, cells were harvested and seeded in 384-well plates (7 μL, 13,000 cells per well), and incubated with increasing concentrations of agonist at 37 °C for 1 h. After incubation, 3 μL d2-labeled IP1 and 3 μL cryptate-labeled anti-IP1 antibody (1:20 diluted in lysis and detection buffer) were added to each well. The fluorescence signal was measured by a CLARIOstar microplate reader with excitation at 330 nm and emission at 620 nm and 665 nm. The data were analyzed using GraphPad Prism software 9.0.
4
Quantitative Measurement of Inositol Phosphate Accumulation
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The IP-One assay kit (Cisbio) was used for the direct quantitative measurement of inositol phosphate (IP) accumulation in CHO-rM1, CHO-rM3 or CHO-rM5 cells, as described previously [17 (link)]. The CHO-hCB1 pERK1/2 assays were performed as described in ref 24 (link).
5
Cellular Signaling Pathway Analysis
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Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin/streptomycin and Lipofectamine 2000® were sourced from Thermo Fisher Scientific (Scoresby, Australia). Foetal Bovine Serum (FBS) was sourced from GE Life Sciences (Parramatta, Australia). The IP-One assay kit was purchased from CisBio (Codolet, France). Atosiban was purchased from Sigma Aldrich (Merck; Sydney, Australia).
6
Quantifying RAGE-Mediated IP1 Signaling
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Cells were seeded at a density of 8000 cells per well onto 96-well transparent cell culture plates and incubated overnight at 37 °C. The following day, cultures were transferred to serum-free conditions and further incubated for 24 h. Thereafter, the cells were treated for 1 h with IP1 stimulation buffer mixed with the same amount of DMEM without phenol including vehicle, RAGE-BSA, HMGB-1, and angiotensin II at an indicated concentration at 37 °C, 5% CO2. Triton X was then added at a final concentration of 1%, and cell lysates were prepared after shaking the plates for 30 min. Finally, the cell lysates were transferred to 384-well white plates and IP1 levels were measured using the IP-One assay kit (Cisbio, France).The emission signals were measured at 620 and 665 nm after excitation at 320 nm, using the ARTEMIS plate reader(Furuno Electric Co. Ltd., Japan).
7
Quantifying IP1 Production in Cells
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The IP-One assay kit (Cisbio) was used for the direct quantitative measurement of myo-Inositol 1 phosphate (IP1). Cells were seeded at 25,000 per well into 96-well transparent cell culture plates and incubated overnight at 37 °C. The following day, cells were pre-incubated with IP1 stimulation buffer (1 mM CaCl2, 0.5 mM MgCl2, 4.2 mM KCl, 146 mM NaCl, 5.5 mM D-Glucose, 10 mM HEPES and 50 mM LiCl, pH 7.4) for 1 h before stimulation with ACh in the presence or absence of increasing concentrations of PAMs in IP1 stimulation buffer for 1 h at 37 °C, 5% CO2. Cells were then lysed with IP1 lysis buffer (50 mM HEPES pH 7.0, 15 mM KF, 1.5% V/V Triton-X-100, 3% V/V FBS, 0.2% W/V BSA), and IP1 levels were measured by incubation of cell lysates with FRET reagents (the cryptate-labeled anti-IP1 antibody and the d2-labeled IP1 analogue) for 1 h at 37 °C. The emission signals were measured at 590 and 665 nm after excitation at 340 nm, using the Envision plate reader (PerkinElmer Life Sciences, Boston, MA). Signals were expressed as the FRET ratio: F = (fluorescence665 nm/fluorescence590 nm) × 104, and normalized to the response to maximal ACh concentration (100 µM).
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