A label-free impedance-based whole cell assay revealed a new G protein-coupled receptor ligand for mouse microglial cell migration

作者：Fukano Yasufumi^*; Okino Nozomu; Furuya Shigeki; Ito Makoto^*

来源：Biochemical and Biophysical Research Communications, 2016, 478(2): 624-630.

DOI：10.1016/j.bbrc.2016.07.119

• G protein-coupled receptor (GPCR)
• Microglia
• Migration
• Impedance-based cell assay
• 5-Oxo-ETE

We report the usefulness of an impedance-based label-free whole cell assay to identify new ligands for G protein-coupled receptors (GPCRs) involved in microglial cell migration. Authentic GPCR ligands were subjected to the impedance-based cell assay in order to examine the responses of ligands for MG5 mouse microglial cells. Complement component 5 (C5a), adenosine 5'-diphosphate (ADP), uridine 5'-triphosphate (UTP), lysophosphatidic acid (LPA), and lysophosphatidylserine (LysoPS) were found to elicit different cellular impedance patterns, i.e. C5a, ADP, and UTP caused a transient increase in cellular impedance, while LPA and LysoPS decreased it. The responses for C5a and ADP were abolished by pertussis toxin (FIX), but not rho-associated protein kinase inhibitor, Y-27632, indicating that C5a and ADP elicited responses through the G alpha i pathway. On the other hand, the response for UTP, LPA or LysoPS was not cancelled by PTX or Y-27632. In a modified Boyden chamber assay, C5a and ADP, but not UTP, LPA, or LysoPS, induced the migration of MG5 cells. These results suggest that PTX-sensitive increase in cellular impedance with the assay is characteristic for ligands of GPCRs involved in microglial cell migration. We found using this assay that 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a new chemoattractant inducing microglial cell migration through the activation of G alpha i.

• 出版日期2016-9-16