The determination of IP1 accumulation was performed using the IP-One HTRF assay (CisBio Bioassays, Bagnol sur Ceze, France), as described previously [45 (link)]. Briefly, cells were transfected and seeded into white 96-well plates. 48 h post-transfection cell media was replaced with 50 μl stimulation buffer containing agonists as indicated. After a 30 min incubation at 37°C, 5% CO2, cells were lysed with 12.5 μl of the supplied conjugate-lysis buffer containing d2-labeled IP1. This was immediately followed by addition of 12.5 μl of conjugate-lysis buffer containing terbium cryptate-labeled anti-IP1 antibody. Following a 1 h incubation at room temperature, fluorescence was measured at 620 and 665 nm 50 μs after excitation at 337 nm using an EnVision 2102 plate reader (PerkinElmer).
Ip one htrf assay
Manufactured by PerkinElmer
Sourced in France
The IP-One HTRF assay is a kit for measuring inositol monophosphate (IP1) levels in cells. It utilizes Homogeneous Time-Resolved Fluorescence (HTRF) technology to quantify IP1, which is a downstream metabolite of the inositol phosphate signaling pathway.
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3 protocols using ip one htrf assay
1
IP1 Accumulation Assay with HTRF
2
HTRF Assay for IP1 Accumulation
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The determination of IP1 accumulation was performed in a 96-well microplate seeded with transfected HEK293FT cells (500 ng/well of a 6-well plate) using the IP-One HTRF assay (CisBio Bioassays) (18 (link), 27 (link)). To construct concentration-response curves, cells expressing either wild-type V2R/Nluc or L312S V2R/Nluc were incubated for 30 minutes at 37°C in the stimulation buffer (10mM HEPES [pH 7.4], 1mM CaCl2, 0.5mM MgCl2, 4mM KCl, 146mM NaCl, 5.5mM glucose, and 50mM LiCl) with or without 1pM–10μM AVP or carbachol (2mM) as an internal control. Cells were then lysed using the conjugate-lysis buffer mixed with the d2-labeled IP1 analog and the terbium cryptate-labeled anti-IP1 antibody according to the manufacturer's instructions. The assay was incubated for 1 hour at room temperature before fluorescence signals were measured at 620 and 665 nm, 50 microseconds after excitation at 337 nm using a CLARIOstar multilabel plate reader.
3
Measuring PAR2-Mediated Gq Signaling
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To determine the effect of PAR2 activation on Gq stimulation, we measured IP1, a downstream metabolite of IP3, using the IP-One HTRF assay from Cis Bio, Medford, MA. HeLa cells were transfected with salmon sperm DNA or PAR2 cDNA as described above, plated in 10 cm culture dishes and incubated at 37°C with 5% CO2. After 24 hours, the cells were trypsinized, counted, plated into 96-well plates at 80,000 cells/well and incubated for another 24 hours. The medium was replaced with a stimulation buffer containing LiCl, per the instructions of the manufacturer. Ligands, including cathepsin S (1 µM), KVDGTS (100 µM) and SLIGRL (10 µM) were added to wells and incubated at 37°C for 1 hour and IP1 levels determined. This assay was performed on multiple wells containing each ligand on three occasions. Error bars represent SEM.
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