Multiplexing High‐Content Flow (HCF) and Quantitative High‐Throughput Screening (qHTS) to Identify Compounds Capable of Decreasing Cell Viability, Activating Caspase 3/7, Expressing Annexin V, and Changing Mitochondrial Membrane Integrity

Lesley A. Mathews1, Jonathan M. Keller1, Crystal McKnight1, Sam Michael1, Paul Shinn1, Dongbo Liu1, Louis M. Staudt2, Craig J. Thomas1, Marc Ferrer1

1 National Institutes of Health, Bethesda, Maryland, 2 National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130060
Online Posting Date:  October, 2013
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Abstract

High‐content flow (HCF) screening systems, such as the iQue Screener and HTFC Screening System from IntelliCyt, have facilitated the implementation of flow cytometry assays for high‐throughput screening. HCF screening systems enable the use of smaller sample volumes and multiplexed assays to simultaneously assess different cellular parameters from a single well. This becomes invaluable when working with cells or compounds that are available in limited quantities or when conducting large‐scale screens. When assays can be miniaturized to a 384‐ or 1536‐well microplate format, it is possible to implement dose‐response‐based high‐throughput screens, also known as quantitative HTS or qHTS. This article describes how qHTS at the new National Center for Advancing Translational Science (NCATS) has been systematically coupled with the HTFC Screening System and Multimetric Apoptosis Screening Kit from IntelliCyt to biologically validate active compounds from primary cell proliferation screens using a model of diffuse large B cell lymphoma (DLBCL). Curr. Protoc. Chem. Biol. 5:195‐212 © 2013 by John Wiley & Sons, Inc.

Keywords: high‐content flow (HCF); quantitative high‐throughput screening (qHTS); cell viability; caspase 3/7; annexin V; mitochondrial membrane integrity

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Assembly of Custom 384‐Well V‐Bottom Compound Plates for Quantitative High‐Throughput Screening (qHTS) Using the Acoustic Transfer System (ATS) from EDC Biosystems
  • Basic Protocol 2: Quantitative High‐Throughput Screening (qHTS) on the HTFC Screening System to Identify Compounds Capable of Activating Caspase 3/7, Expressing Annexin V, and Changing Mitochondrial Membrane Integrity
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Assembly of Custom 384‐Well V‐Bottom Compound Plates for Quantitative High‐Throughput Screening (qHTS) Using the Acoustic Transfer System (ATS) from EDC Biosystems

  Materials
  • Bortezomib (Selleck)
  • Doxorubicin (Biomol)
  • Plinabulin (Selleck)
  • Carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP; Sigma)
  • Staurosporine (IntelliCyt)
  • 100% DMSO (Fisher)
  • Matrix Wellmate
  • Perkin‐Elmer Janus with disposable Varispan tips
  • 384‐well polypropylene plates (Greiner, cat. no. 784201)
  • Benchtop centrifuge for plates (Thermo)
  • 384‐well Acoustic uClear well flat clear‐bottom black tissue‐culture‐treated plates (Greiner, cat. no. 781986)
  • Evolution P3 (EP3) liquid handler with 384MDT head (Perkin Elmer)
  • EDC Biosystems ATS‐100 acoustic dispenser
  • 384‐well V‐bottom polypropylene plates (Greiner, cat. no. 781280)

Basic Protocol 2: Quantitative High‐Throughput Screening (qHTS) on the HTFC Screening System to Identify Compounds Capable of Activating Caspase 3/7, Expressing Annexin V, and Changing Mitochondrial Membrane Integrity

  Materials
  • TMD8 cells in logarithmic growth phase in sterile vented‐cap T225 flasks
  • RPMI medium supplemented with GlutaMAX, 10% FBS, and 1× penicillin/streptomycin (all from Gibco)
  • 70% ethanol
  • Sterile water
  • Pre‐plated 384‐well V‐bottom compound plates or flat‐bottom 384‐well tissue culture plates ( protocol 1)
  • 10% bleach
  • Multimetric No‐Wash Apoptosis Screening kit (IntelliCyt, cat. no. 90053)
    • Caspase detection reagent
    • Annexin V detection reagent
    • Viability reagent
    • Mitochondrial detection reagent
    • 10× binding buffer
  • 1× PBS containing 0.1% BSA
  • Evos f1 inverted microscope with 10× lens and bright‐field filter for visual inspection of cells
  • Cellometer T4 with SD100 counting chambers, for counting cells
  • Thermo Multidrop dispenser with small cassette
  • Sterile stainless‐steel long‐term storage lids (Kalypsys)
  • Cell culture incubator with temperature, CO 2, and relative humidity control
  • HTFC Screening System with bacteriostatic solution (cat. no. 90078), decontamination solution (cat. no. 90077), and cleaning solution (cat. no. 90079) (IntelliCyt)
  • ForeCyt software (preloaded with HTFC Screening System)
  • 8‐ and 6‐bead calibration kit from IntelliCyt (cat. nos. 10162 and 10163)
  • 5‐ml polystyrene round‐bottom tubes (BD Falcon, cat. no. 352058)
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Figures

Videos

Literature Cited

  Burchiel, S.W., Lauer, F.T., Gurule, D., Mounho, B.J., and Salas, V.M. 1999. Uses and future applications of flow cytometry in immunotoxicity testing. Methods 19:28‐35.
  Collins, F.S. 2011. Reengineering translational science: The time is right. Sci. Transl. Med. 3:90cm17.
  Fabian, A., Barok, M., Vereb, G., and Szollosi, J. 2009. Die hard: Are cancer stem cells the Bruce Willises of tumor biology? Cytometry A 75:67‐74.
  Gao, C., Hollingsworth, R.E., and Hurt, E.M. 2012. Introduction to cancer stem cells. In DNA Repair of Cancer Stem Cells, 2013 ed. (L.A. Mathews, S.M. Cabarcas, and E.M. Hurt, eds.) pp. 1‐18. Springer, New York.
  Inglese, J., Auld, D.S., Jadhav, A., Johnson, R.L., Simeonov, A., Yasgar, A., Zheng, W., and Austin, C.P. 2006. Quantitative high‐throughput screening: A titration‐based approach that efficiently identifies biological activities in large chemical libraries. Proc. Natl. Acad. Sci. U.S.A. 103:11473‐11478.
  Inglese, J., Johnson, R.L., Simeonov, A., Xia, M., Zheng, W., Austin, C.P., and Auld, D.S. 2007. High‐throughput screening assays for the identification of chemical probes. Nat. Chem. Biol. 3:466‐479.
  Jia, L., Gopinathan, G., Sukumar, J.T., and Gribben, J.G. 2012. Blocking autophagy prevents bortezomib‐induced NF‐κB activation by reducing I‐κBα degradation in lymphoma cells. PLoS One 7:e32584.
  Luu, Y.K., Rana, P., Duensing, T.D., Black, C., and Will, Y. 2012. Profiling of toxicity and identification of distinct apoptosis profiles using a 384‐well high‐throughput flow cytometry screening platform. J. Biomol. Screen. 17:806‐812.
  Mathews, L., Guha, R., Shinn, P., Lim, K.H., Young, R.M., Keller, J., Liu, D., Goldlust, I.S., Yasgar, A., McKnight, C., Boxer, M.B., Duveau, D.Y., Jiang, J., Michael, S., Mierzwa, T., Huang, W., Walsh, M.J., Mott, B.T., Patel, P.R., Leister, W., Maloney, D.J., LeClair, C.A., Rai, G., Jadhav, A., Peyser, B.D., Austin, C.P., Martin, S., Simeonov, A., Ferrer, M., Staudt, L.M., and Thomas, C.J. High‐throughput combination screening identifies drug‐drug pairings for the Bruton's tyrosine kinase inhibitor ibrutinib against diffuse large B‐cell lymphoma. Submitted for publication.
  Schatton, T., Frank, N.Y., and Frank, M.H. 2009. Identification and targeting of cancer stem cells. Bioessays 31:1038‐1049.
  Sengupta, A. and Cancelas, J.A. 2010. Cancer stem cells: A stride towards cancer cure? J. Cell Physiol. 225:7‐14.
  Shui, W., Yin, L., Luo, J., Li, R., Zhang, W., Zhang, J., Huang, W., Hu, N., Liang, X., Deng, Z.L., Hu, Z., Shi, L.L., Luu, H.H., Haydon, R.C., He, T.C., and Ho, S.H. 2013. Characterization of chondrocyte scaffold carriers for cell‐based gene therapy in articular cartilage repair. J. Biomed. Mater. Res. A doi: 10.1002/jbm.a.34661.
  Telford, W.G., Hawley, T., Subach, F., Verkhusha, V., and Hawley, R.G. 2012. Flow cytometry of fluorescent proteins. Methods 57:318‐330.
  Wang, X., Cui, J., Zhang, B.Q., Zhang, H., Bi, Y., Kang, Q., Wang, N., Bie, P., Yang, Z., Wang, H., Liu, X., Haydon, R.C., Luu, H.H., Tang, N., Dong, J., and He, T.C. 2013. Decellularized liver scaffolds effectively support the proliferation and differentiation of mouse fetal hepatic progenitors. J. Biomed. Mater. Res. A doi: 10.1002/jbm.a.34764.
  Wheat, L.M., Kohlhaas, S.L., Monbaliu, J., De Coster, R., Majid, A., Walewska, R.J., and Dyer, M.J. 2006. Inhibition of bortezomib‐induced apoptosis by red blood cell uptake. Leukemia 20:1646‐1649.
  Wilson, H., Huelsmeyer, M., Chun, R., Young, K.M., Friedrichs, K., and Argyle, D.J. 2008. Isolation and characterisation of cancer stem cells from canine osteosarcoma. Vet. J. 175:69‐75.
  Wlodkowic, D., Skommer, J., and Darzynkiewicz, Z. 2009. Flow cytometry‐based apoptosis detection. Methods Mol. Biol. 559:19‐32.
  Yang, Y., Shaffer, A.L., 3rd, Emre, N.C., Ceribelli, M., Zhang, M., Wright, G., Xiao, W., Powell, J., Platig, J., Kohlhammer, H., Young, R.M., Zhao, H., Xu, W., Buggy, J.J., Balasubramanian, S., Mathews, L.A., Shinn, P., Guha, R., Ferrer, M., Thomas, C., Waldmann, T.A., and Staudt, L.M. 2012. Exploiting synthetic lethality for the therapy of ABC diffuse large B cell lymphoma. Cancer Cell 21:723‐737.
Key References
  Luu et al., 2012. See above.
  Inglese et al., 2006. See above.
Internet Resources
  http://www.intellicyt.com/
  Web site for HTFC products and reagents.
  http://www.ncats.nih.gov/research/reengineering/ncgc/ncgc.html
  Web site for information about screening at NCATS.
  http://www.ncbi.nlm.nih.gov/books/NBK53196/
  Assay Guidance Manual for Screening at NCATS.
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