Applying label-free dynamic mass redistribution assay for studying endogenous FPR1 receptor signalling in human neutrophils

J Pharmacol Toxicol Methods. 2017 Nov;88(Pt 1):72-78. doi: 10.1016/j.vascn.2017.07.003. Epub 2017 Jul 15.

Abstract

Introduction: The label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors (FPRs) in human neutrophils.

Methods: Neutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist potencies.

Results: The potencies (pEC50) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H.

Discussion: We conclude that the DMR assay can be used, and complements more traditional methods, to study the signalling and pharmacology of endogenous FPR receptors in human neutrophils.

Keywords: Dynamic mass redistribution; FPR1; FPR2; GPCR; Label-free assay; Neutrophil; Potency; Signalling.

MeSH terms

  • Biological Assay / methods*
  • Biosensing Techniques / methods*
  • Cell Separation / methods
  • Humans
  • Neutrophils / drug effects
  • Neutrophils / metabolism*
  • Oligopeptides / pharmacology
  • Receptors, Formyl Peptide / antagonists & inhibitors
  • Receptors, Formyl Peptide / metabolism*
  • Signal Transduction / drug effects*

Substances

  • FPR1 protein, human
  • Oligopeptides
  • Receptors, Formyl Peptide
  • Trp-Lys-Tyr-Met-Val-Met