Flow Cytometry Multiplexed Screening Methodologies

doi:10.1017/CBO9781139021500.022

Chapter 17 - Flow Cytometry Multiplexed Screening Methodologies

from Section Four - Chemical Genomics Assays and Screens

Published online by Cambridge University Press: 05 June 2012

Virginia M. Salas ,

J. Jacob Strouse ,

Zurab Surviladze ,

Irena Ivnitski-Steele ,

Bruce S. Edwards and

Larry A. Sklar

Edited by

Haian Fu

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Haian Fu: Affiliation:
Emory University, Atlanta

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Summary

High-content analytical methodologies are increasingly becoming integrated into the screening phase of the discovery pipeline for small molecule modulators of biological targets. The iterative analysis among targets with related functions or among a series of interacting targets can be replaced with multiplexing approaches whereby multiple targets are analyzed simultaneously in the same well against the same compound or even against a mixture of compounds in the same well. It has become practical to use the multiparameter optical capabilities of the flow cytometer for the discovery of active small molecules from compound libraries by high-throughput screening (HTS). In this chapter, we present the fundamental principles of flow cytometry–based multiplex methods, including assay design and data analysis, with specific examples of cell-based and bead-based assays and a discussion of the added value of multiplex data sets.

Overview

Over the past two decades, HTS methodologies (generally defined as testing ten thousand to one hundred thousand compounds per day, depending on the technology) have become an essential part of discovery science for novel drugs and biological probes within the pharmaceutical, biotech, and academic research communities [1–3]. Technologies that are inherently capable of multiparametric measurements, including microscopy and flow cytometry, are well suited to produce high-content, high-complexity data by obtaining multiple data points from each well in a microtiter plate or other high-density format. In multiplex applications, information content is further enriched by obtaining data for each parameter on multiple targets. The advantages, compared with single-point iterative assays, include a substantial savings in time and reagents and the ability to produce rich data sets under conditions that can be optimized for statistical rigor and data quality for multiple targets simultaneously. For example, multiplex analysis among a family of related targets, which often exhibit similar biochemical and pharmacologic properties, produces selectivity screens in a single well with inherent consistency. Furthermore, the conditions in each well are normalized across targets for any number of parameters, and counterscreens for artifacts can be rigorously examined. Table 17.1 is a comparison of characteristics between single-target and multiplexed assays that summarizes the advantages for each approach.

Information

Type: Chapter
Information: Chemical Genomics , pp. 232 - 244

DOI: https://doi.org/10.1017/CBO9781139021500.022 [Opens in a new window]

Publisher: Cambridge University Press

Print publication year: 2012

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References

Pereira, D. AWilliams, J. A 2007 Review: Origin and evolution of high throughput screeningBr J Pharmacol 152 53Google Scholar

Macarron, R 2006 Critical review of the role of HTS in drug discoveryDrug Discov Today 11 277Google Scholar

Bender, ABojanic, DDavies, J. WCrisman, T. JMikhailov, DScheiber, JJenkins, J. LDeng, ZHill, W. APopov, MJacxoby, EGlick, M 2008 Which aspects of HTS are empirically correlated with downstream success?Curr Opin Drug Discov Devel 11 327Google Scholar

Rausch, O 2006 High content cellular screeningCurr Opin Chem Biol 10 316Google Scholar

Lang, PYeow, KNichols, AScheer, A 2006 Cellular imaging in drug discoveryNat Rev Drug Discov 5 343Google Scholar

Korn, KKrausz, E 2007 Cell-based high-content screening of small-molecule librariesCurr Opin Chem Biol 11 503Google Scholar

Bertelsen, M 2006 Multiplex analysis of inflammatory signaling pathways using a high-content imaging systemMethods Enzymol 414 348Google Scholar

Ross, D. ALee, SReiser, VXue, JAlves, KVaidya, SKreamer, AMull, RHudak, EHare, TDetmers, P.ALingham, RFerrer, MStrulovici, BSantini, F 2008 Multiplexed assays by high-content imaging for assessment of GPCR activityJ Biomol Screen 13 449Google Scholar

Irish, J. MHovland, RKrutzik, P. OPerez, O. DBruserud, OGjertsen, B. TNolan, G. P 2004 Single cell profiling of potentiated phosphorylation networks in cancer cellsCell 118 217Google Scholar

Sklar, L. ACarter, M. BEdwards, B. S 2007 Flow cytometry for drug discovery, receptor pharmacology and high-throughput screeningCurr Opin Pharmacol 7 527Google Scholar

Ryningen, ABruserud, O 2007 Epigenetic targeting in acute myeloid leukemia: use of flow cytometry in monitoring therapeutic effectsCurr Pharm Biotechnol 6 401Google Scholar

Kellar, K. LIannone, M. A 2002 Multiplexed microsphere-based flow cytometric assaysExp Hematol 30 1227Google Scholar

Larsen, C. H 2008 The fragile environments of inexpensive CD4+ T-cell enumeration in the last developed countries: strategies for accessible supportCytometry B Clin Cytom 74S 107Google Scholar

Pattanapanyasat, KPhuang-Ngern, YLerdwana, SWasinrapee, PSakulploy, NNoulsri, EThepthai, CMcNicholl, J. M 2007 Evaluation of a single-platform microcapillary flow cytometer for enumeration of absolute CD4+ T-lymphocyte counts in HIV-1 infected Thai patientsCytometry B Clin Cytom 72 387Google Scholar

Li, XYmeti, ALunter, BBreukers, CTibbe, A. GTerstappen, L. WGreve, J 2007 CD4+ T lymphocytes enumeration by an easy-to-use single platform image cytometer for HIV monitoring in resource-constrained settingsCytometry B Clin Cytom 72 397Google Scholar

Edwards, B. SOprea, TProssnitz, E. RSklar, L. A 2004 Flow cytometry for high-throughput, high-content screeningCurr Opin Chem Biol 8 392Google Scholar

Smith, R. AGiorgio, T. D 2004 Cell-based screening: a high throughput flow cytometry platform for identification of cell-specific targeting moleculesComb Chem High Throughput Screen 7 141Google Scholar

Salas, V. MEdwards, B. SSklar, L. A 2008 Advances in multiple analyte profilingAdv Clin Chem 45 47Google Scholar

Waller, ASimons, P. CBiggs, S. MEdwards, B. SProssnitz, E. RSklar, L. A 2004 Techniques: GPCR assembly, pharmacology and screening by flow cytometryTrends Pharmacol Sci 25 663Google Scholar

Nolan, J. PMandy, F 2006 Multiplexed and microparticle-based analyses: quantitative tools for the large-scale analysis of biological systemsCytometry A 69 318Google Scholar

Mandy, F. FNakamura, TBergeron, MSekiguchi, K 2001 Overview and application of suspension array technologyClin Lab Med 4 713Google Scholar

Ayriss, JWoods, TBradbury, APavlik, P 2007 High-throughput screening of single chain antibodies using multiplexed flow cytometryJ Proteome Res 6 1072Google Scholar

Yang, LNolan, J. P 2007 High-throughput screening and characterization of clones selected from phage display librariesCytometry A 71 625Google Scholar

Dunbar, S. A 2006 Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detectionClin Chim Acta 363 71Google Scholar

Nolan, J. PYang, L 2007 The flow of cytometry into systems biologyBrief Funct Genomic Proteomic 6 81Google Scholar

Durr, ODuval, FNichols, ALang, PBrodte, AHeyse, SBesson, D 2007 Robust hit identification by quality assurance and multivariate data analysis of a high-content, cell-based assayJ Biomol Screen 12 1042Google Scholar

Dunlay, R. TCzekalaski, W. JCollins, M. A 2007 Overview of informatics for high content screeningMethods Mol Biol 356 269Google Scholar

Edwards, B. SOprea, TProssnitz, E. RSklar, L. A 2004 Flow cytometry for high-throughput, high-content screeningCurr Opin Chem Biol 8 392Google Scholar

Sklar, L. ACarter, M. BEdwards, B. S 2007 Flow cytometry for drug discovery, receptor pharmacology and high-throughput screeningCurr Opin Pharmacol 7 527Google Scholar

Makarenkov, VZentilli, PKevorkov, DGagarin, AMalo, NNadon, R 2007 An efficient method for the detection and elimination of systematic error in high-throughput screeningBioinformatics 23 1648Google Scholar

Haggerty, S. J 2005 The principle of complementarity: chemical versus biological spaceCurr Opin Chem Biol 9 296Google Scholar

Rosania, G. RCrippen, GWoolf, PStates, DShedden, K 2007 A cheminformatic toolkit for mining biomedical knowledgePharm Res 24 1791Google Scholar

Harris, C. JStevens, A. P 2006 Chemogenomics: structuring the drug discovery process to gene familiesDrug Discov Today 11 880Google Scholar

Noble, M. EEndicott, J. AJohnson, L. N 2004 Protein kinase inhibitors: insights into drug design from structureScience 303 1800Google Scholar

Cherry, MWilliams, D. H 2004 Recent kinase and kinase inhibitor x-ray structures: mechanisms of inhibition and selectivity insightsCurr Med Chem 11 663Google Scholar

Kristiansen, K 2004 Molecular mechanisms of ligand binding, signaling and regulation within G-protein-coupled receptors: molecular modeling mutagenesis approaches to receptor structure and functionPharmacol Ther 103 21Google Scholar

Jacob, LVert, J. P 2008 Protein-ligand interaction prediction: an improved chemogenomics approachBioinformatics 24 2149Google Scholar

Klabunde, TEvers, A 2005 GPCR antitarget modeling: pharmacophore models for biogenic amine binding GPCRs to avoid GPCR-mediated side effectsChembiochem 6 876Google Scholar

Low, JStancato, LLee, JSutherland, J. J 2008 Prioritizing hits from phenotypic high-content screensCurr Opin Drug Discov Devel 11 338Google Scholar

Young, D. WBender, AHoyt, JMcWhinnie, EChirn, G. WTao, C. YTallarico, J. ALabow, MJenkins, J. LMitchison, T. JFeng, Y 2008 Integrating high-content screening and ligand target prediction to identify mechanism of actionNat Chem Biol 4 59Google Scholar

Ross-Macdonald, P 2005 Forward in reverse: how reverse genetics complements chemical geneticsPharmacogenomics 6 429Google Scholar

Bol, DEbner, R 2006 Gene expression profiling in the discovery, optimization and development of novel drugs: one universal screening platformPharmacogenomics 7 227Google Scholar

Kubinyi, H 2006 Chemogenomics in drug discoveryErnst Schering Res Found Workshop 58 1Google Scholar

Waller, ASimons, P. CBiggs, S. MEdwards, B. SProssnitz, E. RSklar, L. A 2004 Techniques: GPCR assembly, pharmacology and screening by flow cytometryTrends Pharmacol Sci 25 663Google Scholar

Ivnitski-Steele, ILarson, R. SLovato, D. MKhawaja, H. MWinter, S. SOprea, T. ISklar, L. AEdwards, B. S 2008 High-throughput flow cytometry to detect selective inhibitors of ABCB1, ABCC1, and ABCG2 transportersAssay Drug Dev Technol 2 263Google Scholar

Shapiro, H. M 2005 Fluorescent probesFlow Cytometry for BiotechnologySklar, L. ANew YorkOxford University Press15

Patsenker, LTatarets, AKolosova, OObukhova, OPovrosin, YFedyunyayeva, IYermolenko, ITerpetschnig, E 2008 Fluorescent probes and labels for biomedical applicationsAnn NY Acad Sci 1130 179Google Scholar

Chen, W 2008 Nanoparticle fluorescence based technology for biological applicationsNanosci Nanotechnol 8 1019Google Scholar

Inglese, JJohnson, R. LSimeonov, AXia, MZheng, WAustin, C. PAuld, D. S 2007 High-throughput screening assays for the identification of chemical probesNat Chem Biol 3 466Google Scholar

Mayr, L. MFuerst, P 2008 The future of high-throughput screeningJ Biomol Screen 13 443Google Scholar

Ling, X. B 2008 High throughput screening informaticsComb Chem High Throughput Screen 11 249Google Scholar

Harper, GPickett, S. D 2008 Methods for mining HTS dataDrug Discov Today 11 694Google Scholar

Dunlay, R. TCzekalaski, W. JCollins, M. A 2007 Overview of informatics for high content screeningMethods Mol Biol 356 269Google Scholar

Garfinkle, L. S 2007 Large-scale data management for high content screeningMethods Mol Biol 356 281Google Scholar

Krutzik, PNolan, G 2006 Fluorescent cell barcoding allows high throughput drug screening by flow cytometryNat Meth 3 361Google Scholar

Nolan, J. PSklar, L. A 2002 Suspension array technology; evolution of the flat array paradigmTIBS 20 9Google Scholar

Schwartz, S. LTessema, MBuranda, TPylypenko, ORak, ASimons, P. CSurviladze, SSklar, L.AWandinger-Ness, A 2008 Flow cytometry for real-time measurement of guanine nucleotide binding and exchange by Ras-like GTPasesAnal Biochem 381 258Google Scholar

Roman, D. LTalbot, J. NRoof, R. ASunahara, R. KTraynor, J. RNeubig, R. R 2007 Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assayMol Pharmacol 71 169Google Scholar

Nolan, J. PSklar, L. A 1998 The emergence of flow cytometry for the sensitive real-time analysis of molecular assemblyNat Biotechnol 16 833Google Scholar

Hanley, B 2007 Variance in multiplex suspension array assays: carryover of microspheres between sample wellsJ Negat Results Biomed 6 6Google Scholar

Hanley, B 2007 Variance in multiplex suspension array assays: intraplex method improves reliabilityTheor Biol Med Model 4 32Google Scholar

Hanley, BXing, LCheng, R. H 2007 Variance in multiplex suspension array assays: microsphere size variation impactTheor Biol Med Model 4 31Google Scholar

Zhang, JChung, T. DOldenburg, K. R 1999 A simple statistical parameter for use in evaluation and validation of high throughput screening assaysJ Biomol Screen 4 67Google Scholar

Glickman, J. FAuld, D 2005 Another Z factorAssay Drug Devel Technol 3 339Google Scholar

Malo, NHanley, J. ACerquozzi, SPelletier, JNadon, R 2006 Statistical practice in high-throughput screening data analysisNat Biotechnol 24 167Google Scholar

Young, S. MBologa, C. MFara, DBryant, B.KStrouse, J. JArterburn, J. BYe, R. DOprea, T. IProssnitz, E. RSklar, L. AEdwards, B.S 2008 Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probesCytometry A 75 253Google Scholar

Ivnitski-Steele, ILarson, R. SLovato, D. MKhawaja, H. MWinter, S. SOprea, T. ISklar, L. AEdwards, B.S 2008 High-throughput flow cytometry to detect selective inhibitors of ABCB1, ABCC1, and ABCG2 transportersAssay Drug Dev Technol 2 263Google Scholar

Curpan, R. FSimons, P. CZhai, DYoung, S. MCarter, M. BBologa, C.GOprea, T. ISatterthwait, A. CReed, J. CEdwards, B.SSkklar, L. A 2011 High-throughput screen for the chemical inhibitors of anti-apoptotic Bcl-2 family proteins by multiplex flow cytometryAssay Drug Dev TechnolE-pub ahead of print

McEwen, D. PGee, K. RKang, H. CNeubig, R 2001 Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteinsAnal Biochem 291 109Google Scholar

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