Cell-based no-wash fluorescence assays for compound screens using a fluorescence cytometry plate reader

The Journal of Pharmacology and Experimental Therapeutics
Kirill GorshkovWei Zheng

Abstract

High-throughput cell-based fluorescent imaging assays often require removal of background fluorescent signal to obtain robust measurements. Processing high density microplates to remove background signal is challenging due to equipment requirements and increasing variation after multiple plate wash steps. Here, we present the development of a wash-free cell-based fluorescence assay method for high-throughput screening (HTS) using a laser scanning fluorescence plate cytometer. The cytometry data consisted of cell count and fluorescent intensity measurements for phenotypic screening. The advantages of this assay format include (1) no plate washing, (2) four-fold faster plate scan and analysis time, (3) high-throughput, and (4) greater than ten-fold smaller and direct data files. In contrast, traditional imaging assays require multiple plate washes to remove the background signal, long plate scan and data analysis times, and large data files. We obtained robust screening results by applying this assay methodology for two lysosomal storage diseases and two viral infections. Therefore, this versatile and broadly applicable Mirrorball-based method greatly improves the throughput and data quality of image-based screening by increasing...Continue Reading

References

Oct 18, 2000·Proceedings of the National Academy of Sciences of the United States of America·X FangG B Mills
Nov 2, 2004·International Journal of Clinical Pharmacology and Therapeutics·A R van TroostenburgP Fishman
May 3, 2007·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Martin J EdelmanJanet Dancey
Jan 28, 2009·Proceedings of the National Academy of Sciences of the United States of America·Benny LiuJohn M Dietschy
Aug 4, 2009·The Oncologist·AeRang KimBrigitte C Widemann
Nov 28, 2009·The EMBO Journal·Nabil DjouderWilhelm Krek
Mar 30, 2010·Trends in Biotechnology·Fabian ZanellaWolfgang Link
Dec 29, 2010·Journal of Cell Science·Yongqiang Chen, Daniel J Klionsky
Jul 14, 2011·Autophagy·Hege UglandHeidi Kiil Blomhoff
Nov 9, 2011·Frontiers in Molecular Neuroscience·Hagit Eldar-Finkelman, Ana Martinez
Dec 12, 2012·Molecular Genetics and Metabolism·Muneaki MatsuoKousaku Ohno
Nov 13, 2013·The Heart Surgery Forum·Arndt-Holger KiesslingArdawan Julian Rastan
Nov 9, 2014·Journal of Biomolecular Screening·Elizabeth EnglandChristine J Rossant
Dec 19, 2014·Frontiers in Pharmacology·Birgit T Priest, Gul Erdemli
Dec 30, 2014·Frontiers in Cellular Neuroscience·Kirill Gorshkov, Jin Zhang
Feb 25, 2015·Assay and Drug Development Technologies·Lynn RasmussenJames W Noah
Feb 7, 2017·Molecular Genetics and Metabolism·Edward H Schuchman, Robert J Desnick
Feb 14, 2017·Nature Chemical Biology·Kirill GorshkovJin Zhang
Jul 19, 2017·Proceedings of the National Academy of Sciences of the United States of America·Angel BalmasedaDavide Corti
Dec 14, 2017·Essays in Biochemistry·Elena SeranovaSovan Sarkar
Jan 31, 2018·Oxidative Medicine and Cellular Longevity·Romina MancinelliClaudia Giampietri
Apr 9, 2019·Assay and Drug Development Technologies·Emily M LeeWei Zheng

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Datasets Mentioned

BETA
GM05659
GM03123
GM13205

Methods Mentioned

BETA
fluorescence microscopy
Assay
transgenic

Clinical Trials Mentioned

NCT02534844

Software Mentioned

Cellista
Excel
Fiji
Graphpad Prism

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