Skip to main content

Advertisement

SpringerLink
Log in

Cell-Based Assays for High-Throughput Screening

  • Review
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Cell-based assays represent approximately half of all high-throughput screens currently performed. Here, we review in brief the history and status of high-throughput screening (HTS), and summarize some of the challenges and benefits associated with the use of cell-based assays in HTS. Approaches for successful experimental design and execution of cell-based screens are introduced, including strategies for assay development, implementation of primary and secondary screens, and target identification. In doing so, we hope to provide a comprehensive review of the cell-based HTS process and an introduction to the methodologies and techniques used.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Hopkins, A. L., & Groom, C. R. (2002). The druggable genome. Nature Reviews Drug Discovery, 1(9), 727–730.

    Article  CAS  Google Scholar 

  2. Beggs, M. (2000). HTS—where next. Drug Discovery World, Winter, 25–30.

  3. Hertzberg, R. P., & Pope, A. J. (2000). High-throughput screening: New technology for the 21st century. Current Opinion in Chemical Biology, 4(4), 445–451.

    Article  CAS  Google Scholar 

  4. Liu, B., Li, S., & Hu, J. (2004). Technological advances in high-throughput screening. American Journal of Pharmacogenomics, 4(4), 263–276.

    Article  CAS  Google Scholar 

  5. Grozinger, K., Proudfoot, J., & Hargrave, K. (2006). Discovery and development of nevirapine. In M. S. Chorghade (Ed.), Drug discovery and development, Volume I: Drug discovery (pp. 353–363). Weinheim: Wiley.

  6. Kell, D. (1999). Screensavers: Trends in high-throughput analysis. Trends in Biotechnology, 17(3), 89.

    Article  CAS  Google Scholar 

  7. Zitzler, J., Link, D., Schafer, R., et al. (2004). High-throughput functional genomics identifies genes that ameliorate toxicity due to oxidative stress in neuronal HT-22 cells: GFPT2 protects cells against peroxide. Molecular & Cellular Proteomics, 3(8), 834–840.

    Article  CAS  Google Scholar 

  8. Korherr, C., Gille, H., Schafer, R., et al. (2006). Identification of proangiogenic genes and pathways by high-throughput functional genomics: TBK1 and the IRF3 pathway. PNAS, 103(11), 4240–4245.

    Article  CAS  Google Scholar 

  9. Moffat, J., Grueneberg, D. A., Yang, X., et al. (2006). A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell, 124(6), 1283–1298.

    Article  CAS  Google Scholar 

  10. Inglese, J., Auld, D. S., Jadhav, A., et al. (2006). Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries. PNAS, 103(31), 11473–11478.

    Article  CAS  Google Scholar 

  11. Tolliday, N., Clemons, P. A., Ferraiolo, P., et al. (2006). Small molecules, big players: The National Cancer Institute’s Initiative for Chemical Genetics. Cancer Research, 66(18), 8935–8942.

    Article  CAS  Google Scholar 

  12. Burns, S., Travers, J., Collins, I., et al. (2006). Identification of small-molecule inhibitors of protein kinase B (PKB/AKT) in an AlphaScreenTM high-throughput screen. Journal of Biomolecular Screening, 11(7), 822–827.

    Article  CAS  Google Scholar 

  13. Sudo, K., Yamaji, K., Kawamura, K., et al. (2005). High-throughput screening of low molecular weight NS3-NS4A protease inhibitors using a fluorescence resonance energy transfer substrate. Antiviral Chemistry & Chemotherapy, 16(6), 385–392.

    CAS  Google Scholar 

  14. Swaney, S., McCroskey, M., Shinabarger, D., et al. (2006). Characterization of a high-throughput screening assay for inhibitors of elongation factor P and ribosomal peptidyl transferase activity. Journal of Biomolecular Screening, 11(7), 736–742.

    Article  CAS  Google Scholar 

  15. Allen, M., Reeves, J., & Mellor, G. (2000). High throughput fluorescence polarization: A homogeneous alternative to radioligand binding for cell surface receptors. Journal of Biomolecular Screening, 5(2), 63–69.

    Article  CAS  Google Scholar 

  16. Xu, J., Wang, X., Ensign, B., et al. (2001). Ion-channel assay technologies: Quo vadis? Drug Discovery Today, 6(24), 1278–1287.

    Article  CAS  Google Scholar 

  17. Parker, G. J., Law, T. L., Lenoch, F. J., et al. (2000). Development of high throughput screening assays using fluorescence polarization: Nuclear receptor-ligand-binding and kinase/phosphatase assays. Journal of Biomolecular Screening, 5(2), 77–88.

    Article  CAS  Google Scholar 

  18. Kenny, C. H., Ding, W., Kelleher, K., et al. (2003). Development of a fluorescence polarization assay to screen for inhibitors of the FtsZ/ZipA interaction. Analytical Biochemistry, 323(2), 224–233.

    Article  CAS  Google Scholar 

  19. Chambers, C., Smith, F., Williams, C., et al. (2003). Measuring intracellular calcium fluxes in high throughput mode. Combinatorial Chemistry & High Throughput Screening, 6(4), 355–362.

    CAS  Google Scholar 

  20. Kariv, I., Stevens, M. E., Behrens, D. L., et al. (1999). High throughput quantitation of cAMP production mediated by activation of seven transmembrane domain receptors. Journal of Biomolecular Screening, 4(1), 27–32.

    Article  CAS  Google Scholar 

  21. Li, X., Shen, F., Zhang, Y., et al. (2007). Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay. European Journal of Pharmaceutics and Biopharmaceutics [Epub ahead of print].

  22. Beck, V., Pfitscher, A., & Jungbauer, A. (2005). GFP-reporter for a high throughput assay to monitor estrogenic compounds. Journal of Biochemical and Biophysical Methods, 64(1), 19–37.

    Article  CAS  Google Scholar 

  23. Yarrow, J. C., Totsukawa, G., Charras, G. T., et al. (2005). Screening for cell migration inhibitors via automated microscopy reveals a rho-kinase inhibitor. Chemistry & Biology, 12(3), 385–395.

    Article  CAS  Google Scholar 

  24. Eggert, U. S., Kiger, A. A., Richter, C., et al. (2004). Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets. PLoS Biology, 2(12), e379.

    Article  Google Scholar 

  25. Krejci, P., Pejchalova, K., & Wilcox, W. R. (2007). Simple, mammalian cell-based assay for identification of inhibitors of the Erk MAP kinase pathway. Investigational New Drugs [Epub ahead of print].

  26. Bradley, J., Gill, J., Bertelli, F., et al. (2004). Development and automation of a 384-well cell fusion assay to identify inhibitors of CCR5/CD4-mediated HIV virus entry. Journal of Biomolecular Screening, 9(6), 516–524.

    Article  CAS  Google Scholar 

  27. Wunder, F., Stasch, J. P., Hutter, J., et al. (2005). A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the nitric oxide/cGMP pathway. Analytical Biochemistry, 339(1), 104–112.

    Article  CAS  Google Scholar 

  28. Brandish, P. E., Chiu, C. S., Schneeweis, J., et al. (2006). A cell-based ultra-high-throughput screening assay for identifying inhibitors of D-amino acid oxidase. Journal of Biomolecular Screening, 11(5), 481–487.

    Article  CAS  Google Scholar 

  29. Barberis, A., Gunde, T., Berset, C., et al. (2005). Yeast as a screening tool. Drug Discovery Today, 2, 187–192.

    Article  CAS  Google Scholar 

  30. Balgi, A. D., & Roberge, M. (2009). Screening for chemical inhibitors of heterologous proteins expressed in yeast using a simple growth-restoration assay. Methods in Molecular Biology, 486, 125–138.

    Article  CAS  Google Scholar 

  31. Puri, A. W., & Bogyo, M. (2009). Using small molecules to dissect mechanisms of microbial pathogenesis. ACS Chemical Biology, 4(8), 603–616.

    Article  CAS  Google Scholar 

  32. Zlitni, S., Blanchard, J. E., & Brown, E. D. (2009). High-throughput screening of model bacteria. Methods in Molecular Biology, 486, 13–28.

    Article  CAS  Google Scholar 

  33. Hong, C. C. (2009). Large-scale small-molecule screening using zebrafish embryos. Methods in Molecular Biology, 486, 43–56.

    Article  CAS  Google Scholar 

  34. Zon, L. I., & Peterson, R. T. (2005). In vivo drug discovery in the zebrafish. Nature Reviews. Drug Discovery, 4(1), 35–44.

    Article  CAS  Google Scholar 

  35. O’Rourke, E. J., Conery, A. L., & Moy, T. I. (2009). Whole-animal high-throughput screens: The C. Elegans model. Methods In Molecular Biology, 486, 57–76.

    Article  Google Scholar 

  36. Moy, T. I., Conery, A. L., Larkins-Ford, J., Wu, G., Mazitschek, R., Casadei, G., et al. (2009). High-throughput screen for novel antimicrobials using a whole animal infection model. ACS Chemical Biology, 4(7), 527–533.

    Article  CAS  Google Scholar 

  37. Kwok, T. C., Ricker, N., Fraser, R., Chan, A. W., Burns, A., Stanley, E. F., et al. (2006). A small-molecule screen in C. Elegans yields a new calcium channel antagonist. Nature, 441(7089), 91–95.

    Article  CAS  Google Scholar 

  38. Agee, A., & Carter, D. (2009). Whole-organism screening: Plants. Methods in Molecular Biology, 486, 77–96.

    Article  CAS  Google Scholar 

  39. Norambuena, L., Raikhel, N. V., & Hicks, G. R. (2009). Chemical genomics approaches in plant biology. Methods in Molecular Biology, 553, 345–354.

    Article  CAS  Google Scholar 

  40. An, F. (2009). Fluorescence-based assays. Methods in Molecular Biology, 486, 97–107.

    Article  CAS  Google Scholar 

  41. Taylor, D. L., Haskins, J. R., & Giuliano, K. A. (Eds.). (2006). High content screening—A powerful approach to systems cell biology and drug discovery. Methods in Molecular Biology (p. 356). Totowa, NJ: Humana Press.

    Google Scholar 

  42. Sklar, L. A., Carter, M. B., & Edwards, B. S. (2007). Flow cytometry for drug discovery, receptor pharmacology and high-throughput screening. Current Opinion in Pharmacology, 7(5), 527–534.

    Article  CAS  Google Scholar 

  43. Edwards, B. S., Young, S. M., Ivnitsky-Steele, I., Ye, R. D., Prossnitz, E. R., & Sklar, L. A. (2009). High-content screening: Flow cytometry analysis. Methods in Molecular Biology, 486, 151–165.

    Article  CAS  Google Scholar 

  44. Zhang, J.-H., Chung, T. D. Y., & Oldenburg, K. R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening, 4(2), 67–73.

    Article  Google Scholar 

  45. Josiah, S. (2009). Interpretation of uniform-well readouts. Methods in Molecular Biology, 486, 177–192.

    Article  CAS  Google Scholar 

  46. Carpenter, A. E. (2009). Extracting rich information from images. Methods in Molecular Biology, 486, 193–211.

    Article  CAS  Google Scholar 

  47. Harding, M. W., Handschumacher, R. E., & Speicher, D. W. (1986). Isolation and amino acid sequence of cyclophilin. The Journal Of Biological Chemistry, 261(18), 8547–8555.

    CAS  Google Scholar 

  48. Handschumacher, R. E., Harding, M. W., Rice, J., et al. (1984). Cyclophilin: A specific cytosolic binding protein for cyclosporin A. Science, 226(4674), 544–547.

    Article  CAS  Google Scholar 

  49. Fischer, G., Wittmann-Liebold, B., Lang, K., et al. (1989). Cyclophilin and peptidyl-prolyl cis–trans isomerase are probably identical proteins. Nature, 337(6206), 476–478.

    Article  CAS  Google Scholar 

  50. Takahashi, N., Hayano, T., & Suzuki, M. (1989). Peptidyl-prolyl cis–trans isomerase is the cyclosporin A-binding protein cyclophilin. Nature, 337(6206), 473–475.

    Article  CAS  Google Scholar 

  51. Lane, W. S., Galat, A., Harding, M. W., et al. (1991). Complete amino acid sequence of the FK506 and rapamycin binding protein, FKBP, isolated from calf thymus. Journal of Protein Chemistry, 10(2), 151–160.

    Article  CAS  Google Scholar 

  52. Harding, M. W., Galat, A., Uehling, D. E., et al. (1989). A receptor for the immunosuppressant FK506 is a cis–trans peptidyl-prolyl isomerase. Nature, 341(6244), 758–760.

    Article  CAS  Google Scholar 

  53. Siekierka, J. J., Hung, S. H. Y., Poe, M., et al. (1989). A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature, 341(6244), 755–757.

    Article  CAS  Google Scholar 

  54. Taunton, J., Hassig, C. A., & Schreiber, S. L. (1996). A mammalian histone deacetylase related to the yeast transcriptional regulator Rpd3p. Science, 272(5260), 408–411.

    Article  CAS  Google Scholar 

  55. Burdine, L., & Kodadek, T. (2004). Target identification in chemical genetics: the (often) missing link. Chemistry & Biology, 11(5), 593–597.

    Article  CAS  Google Scholar 

  56. Rix, U., & Superti-Furga, G. (2009). Target profiling of small molecules by chemical proteomics. Nature Chemical Biology, 5(9), 616–624.

    Article  CAS  Google Scholar 

  57. Colca, J. R., & Harrigan, G. G. (2004). Photo-affinity labeling strategies in identifying the protein ligands of bioactive small molecules: Examples of targeted synthesis of drug analog photoprobes. Combinatorial Chemistry & High Throughput Screening, 7(7), 699–704.

    CAS  Google Scholar 

  58. Kolb, H. C., & Sharpless, K. B. (2003). The growing impact of click chemistry on drug discovery. Drug Discovery Today, 8(24), 1128–1137.

    Article  CAS  Google Scholar 

  59. Speers, A. E., & Cravatt, B. F. (2004). Profiling enzyme activities in vivo using click chemistry methods. Chemistry & Biology, 11(4), 535–546.

    Article  CAS  Google Scholar 

  60. Maly, D. J., Choong, I. C., & Ellman, J. A. (2000). Combinatorial target-guided ligand assembly: Identification of potent subtype-selective c-Src inhibitors. PNAS, 97(6), 2419–2424.

    Article  CAS  Google Scholar 

  61. Sem, D. S., Bertolaet, B., Baker, B., et al. (2004). Systems-based design of bi-ligand inhibitors of oxidoreductases: Filling the chemical proteomic toolbox. Chemistry & Biology, 11(2), 185–194.

    CAS  Google Scholar 

  62. Profit, A. A., Lee, T. R., & Lawrence, D. S. (1999). Bivalent inhibitors of protein tyrosine kinases. Journal of the American Chemical Society, 121(2), 280–283.

    Article  CAS  Google Scholar 

  63. Ong, S. E., Blagoev, B., Kratchmarova, I., et al. (2002). Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Molecular & Cellular Proteomics, 1(5), 376–386.

    Article  CAS  Google Scholar 

  64. Ong, S. E., Schenone, M., Margolin, A. A., Li, X., Do, K., Doud, M. K., et al. (2009). Identifying the proteins to which small-molecule probes and drugs bind in cells. Proceedings of the National Academy of Sciences of the United States of America, 106(12), 4617–4622.

    Article  CAS  Google Scholar 

  65. Lum, P. Y., Armour, C. D., Stepaniants, S. B., et al. (2004). Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes. Cell, 116(1), 121–137.

    Article  CAS  Google Scholar 

  66. Giaever, G., Flaherty, P., Kumm, J., et al. (2004). Chemogenomic profiling: Identifying the functional interactions of small molecules in yeast. PNAS, 101(3), 793–798.

    Article  CAS  Google Scholar 

  67. Luesch, H. (2006). Towards high-throughput characterization of small molecule mechanisms of action. Molecular Biosystems, 2(12), 609–620.

    Article  CAS  Google Scholar 

  68. Kley, N. (2004). Chemical dimerizers and three-hybrid systems: Scanning the proteome for targets of organic small molecules. Chemistry & Biology, 11(5), 599.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Drs. M. Schenone and I. Smukste for insightful discussions on target identification. The study was funded in whole or in part with federal funds from the National Cancer Institute’s Initiative for Chemical Genetics, National Institutes of Health, under Contract no. N01-CO-12400.

Author information

Authors and Affiliations

  1. Chemical Biology Platform, The Broad Institute, 7 Cambridge Center, Cambridge, MA, 02142, USA

    W. Frank An & Nicola Tolliday

Authors
  1. W. Frank An
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nicola Tolliday
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nicola Tolliday.

Rights and permissions

Reprints and permissions

About this article

Cite this article

An, W.F., Tolliday, N. Cell-Based Assays for High-Throughput Screening. Mol Biotechnol 45, 180–186 (2010). https://doi.org/10.1007/s12033-010-9251-z

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-010-9251-z

Keywords

Search

Navigation