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Chapter 17 - Flow Cytometry Multiplexed Screening Methodologies
- from Section Four - Chemical Genomics Assays and Screens
- Edited by Haian Fu, Emory University, Atlanta
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- Book:
- Chemical Genomics
- Published online:
- 05 June 2012
- Print publication:
- 13 February 2012, pp 232-244
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- Chapter
- Export citation
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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.