17 May 2010 A cellular assay using metal-modified fluorescence lifetime analysis for high-content screening of protein internalization
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Abstract
Current high-content screening (HCS) techniques involve the analysis of cellular assays using high-resolution imaging combined with sophisticated algorithms for automated image analysis. Commercially available platforms are invariably highly specialised and expensive. Here we present a novel assay utilising changes in fluorescence lifetime in the vicinity of a rough Au film. A mammary carcinoma cell line was created expressing EGFP in the membrane, and cells were plated onto multi-well slides covered with a 30 nm Au film. FLIM images show a large reduction in lifetime for membrane-bound GFP in close proximity to the Au surface. Addition of a suitable ligand leads to internalization of the GFP with a corresponding increase in lifetime. The degree of internalization can be very quickly and easily checked using standard lifetime analysis techniques, with no need for image analysis. We demonstrate the method by comparing the efficacies of two small molecule inhibitors interfering with the internalization process.
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Nic Cade, Nic Cade, Gilbert Fruhwirth, Gilbert Fruhwirth, Stephen J. Archibald, Stephen J. Archibald, Tony Ng, Tony Ng, David Richards, David Richards, } "A cellular assay using metal-modified fluorescence lifetime analysis for high-content screening of protein internalization", Proc. SPIE 7715, Biophotonics: Photonic Solutions for Better Health Care II, 77150P (17 May 2010); doi: 10.1117/12.852547; https://doi.org/10.1117/12.852547
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