A cellular screening assay using analysis of metal-modified fluorescence lifetime.
Current methods for screening cell receptor internalization often require complex image analysis with limited sensitivity. Here we describe a novel bioassay based on detection of changes in global fluorescence lifetime above a gold substrate, with superresolution axial sensitivity and no need for image analysis. We show that the lifetime of enhanced green fluorescent protein expressed in a cellular membrane is greatly reduced in close proximity to the gold, resulting in a distance-dependent lifetime distribution throughout the cell. We demonstrate the application of this phenomenon in a screening assay by comparing the efficacies of two small molecule inhibitors interfering with the internalization process of a G protein-coupled receptor.
Cade
Nicholas I
NI
Department of Physics, King's College London, London, United Kingdom. nicholas.cade@kcl.ac.uk
Fruhwirth
Gilbert
G
Archibald
Stephen J
SJ
Ng
Tony
T
Richards
David
D
D001681:Biological Assay / Q000379:methods*; D002138:Calibration; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry; D054377:Chemokine CXCL12 / Q000737:chemistry; D003198:Computer Simulation; D004705:Endocytosis; D005453:Fluorescence; D006046:Gold / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation; D019718:Receptors, CXCR4 / Q000737:chemistry / Q000235:genetics; D013997:Time Factors
C515255:CXCL12 protein, human; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D019718:Receptors, CXCR4; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006046:Gold