Reagents and Instruments for Multiplexed Analysis Using Microparticles

John P. Nolan1, Loretta Yang1, Henri C. van der Heyde1

1 La Jolla Bioengineering Institute, La Jolla, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.8
DOI:  10.1002/0471142956.cy1308s37
Online Posting Date:  August, 2006
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Multiplexed molecular analysis by means of flow cytometry using optically encoded microspheres is a rapidly expanding application that has its roots in the earliest days of flow cytometry. The approach is driven by increasing demand for analytical methods to measure large numbers of biomolecules quantitatively and sensitively in small volumes of sample. Encoded microspheres and flow cytometry have been employed for a wide range of multiplexed molecular analysis, and detailed protocols for many of these have been developed. The goal of this unit is to provide an overview of the concepts, instruments, and reagents that enable these assays.

Keywords: flow cytometry, proteomic, genomic, quantitation, molecular

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Table of Contents

  • Background and History
  • Nomenclature
  • Microparticles and Optical Encoding
  • Instrumentation and Software
  • Assays and Kits
  • Concluding Remarks
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Battersby, B.J., Lawrie, G.A., Johnston, A.P., and Trau M. 2002. Optical barcoding of colloidal suspensions: Applications in genomics, proteomics and drug discovery. Chem. Commun. (Camb.) 14:1435‐1441.
   Faucher, S., Martel, A., Sherring, A., Bogdanovic, D., Malloch, L., Kim, J.E., Bergeron, M., Sandstrom, P., and Mandy, F.F. 2006. A combined HIV‐1 protein bead array for serology assay and T‐cell subset immunophenotyping with a hybrid flow cytometer: A step in the direction of a comprehensive multitasking instrument platform for infectious disease diagnosis and monitoring. Cytometry B Clin. Cytom. 70B:179‐188.
   Fenniri, H., Chun S., Ding L.H., Zyrianov Y., and Hallenga, K. 2003. Preparation, physical properties, on‐bead binding assay and spectroscopic reliability of 25 barcoded polystyrene‐poly(ethylene glycol) graft copolymers. J. Am. Chem. Soc. 125:10546‐10560.
   Gao, X. and Nie, S. 2004. Quantum dot–encoded mesoporous beads with high brightness and uniformity: Rapid readout using flow cytometry. Anal. Chem. 76:2406‐2410.
   Han, M., Gao, X., Su, J.Z., and Nie, S. 2001. Quantum‐dot‐tagged microbeads for multiplexed optical coding of biomolecules. Nat. Biotechnol. 19:631‐635.
   Horan, P.K. and Wheeless, L.L. Jr. 1977. Quantitative single cell analysis and sorting. Science 198:149‐157.
   Iannone, M.A. 2001. Microsphere‐based molecular cytometry. Clin. Lab Med. 21:731‐742.
   Janossy, G., Jani, I.V., Kahan, M., Barnett, D., Mandy, F., and Shapiro, H. 2002. Precise CD4 T‐cell counting using red diode laser excitation: For richer, for poorer. Cytometry 50:78‐85.
   Keij, J.F. and Steinkamp, J.A. 1998. Flow cytometric characterization and classification of multiple dual‐color fluorescent microspheres using fluorescence lifetime. Cytometry 33:318‐323.
   Kettman, J.R., Davies, T., Chandler, D., Oliver, K.G., and Fulton, R.J. 1998. Classification and properties of 64 multiplexed microsphere sets. Cytometry 33:234‐243.
   Mandecki, W., Ernst, E., and Kogan, N. 2000. Light‐powered microtransponders for high multiplex‐level analyses of nucleic acids. Abstr. Pap. Am. Chem. Soc. 219:U755.
   Nicewarner‐Pena, S.R., Freeman, R.G., Reiss, B.D., He, L., Pena, D.J., Walton, I.D., Cromer, R., Keating, C.D., and Natan, M.J. 2001. Submicrometer metallic barcodes. Science 294:137‐141.
   Nolan, J.P. and Mandy, F. 2006. Multiplexed and microparticle‐based analyses: Quantitative tools for the large‐scale analysis of biological systems. Cytometry A 69:318‐325.
   Nolan, J.P. and Sklar, L.A.. 2002. Suspension array technology: evolution of the flat‐array paradigm. Trends Biotechnol. 20:9‐12.
   Rodriguez‐Caballero, A., Garcia‐Montero, A.C., Bueno, C., Almeida, J., Varro, R., Chen, R., Pandiella, A., and Orfao, A. 2004. A new simple whole blood flow cytometry–based method for simultaneous identification of activated cells and quantitative evaluation of cytokines released during activation. Lab Invest. 84:1387‐1398.
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