Intracellular IP1 measurement

Introduction

The detection of intracellular second messengers such as IP1, produced following the activation of receptors coupled to Gq proteins, can be achieved by HTRF® technology.
The HTRF® technology (CISBIO) is based on the basic principle of FRET. 
The properties of the fluorophores used provide many advantages.

Principle

This is a competition assay for the binding of an anti-IP1 antibody linked to the donor fluorophore, between the intracellular IP1 and the IP1 provided by the kit fused to the acceptor fluorophore.
LiCl is added to the reaction buffer to allow the accumulation of the IP1 produced in the cells.

Protocol

Format384 wells
Cellular ModelHEK293 in suspension   possibility to work on other lines
Distribution of cells in the plate
Stimulation
Incubation37°C variable time depending on cell type and receptor

Addition of HTRF reagents

Incubation1H at 21°C
DetectionExcitation wavelength : 337 nm
Emission wavelength 1 : 665 nm
emission of the donor fluorophore
Emission wavelength 2 : 615 nm
emission of the acceptor fluorophore

 

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Notes :

  • The quantity of cells expressing the receptor of interest to use in the experiment (transient or stable line) needs to be evaluated during assay validation.
  • Allow 2 to 3 mg of compounds to be tested

Recommendation :

  • Make sure the sample is soluble in a solvent compatible with cell culture

Determination of intracellular IP1 following oxytocin receptor stimulation

Cells  : HEK293 overexpressing the oxytocin receptor

Analysis: S= Signal 665 nm / Signal 615 nm

Measurement of the agonist effect of oxytocin and carbetocin
Measurement of the antagonistic effect of compound L-869,899 in the presence of 30 nM agonist

All models can be adapted to your request.