National Flow Cytometry and Sorting Research Resource

National Flow Cytometry and Sorting Research Resource

Research Emphasis

The resource advances flow cytometric analyses through innovative research, development, and collaborations. Flow cytometry is a technique for high-speed analysis of individual particles ranging in size from single molecules and macromolecular complexes to subcellular organelles, cells, and cellular aggregates. Particles pass rapidly through one or more focused laser beams, where probe molecules bound to specific components, such as DNA in cells, are excited and the emitted fluorescence photons are detected. Measurement of fluorescence emissions and scattered excitation light provides quantitative information about the particles. For cells, measurements are made of DNA, RNA, and protein contents; surface molecules; internal antigens; and physiologic parameters. A wide variety of assay systems based on microspheres (generally 5-10 µm in diameter) are also available, including specific DNA/RNA binding, protein analysis, protein-protein and protein-nucleic acid interactions, and enzymatic activity. Because individual particles are analyzed, distributions of these and other measured parameters are obtained at analysis rates of thousands of events per second. On the basis of these measurements, particles in selected subpopulations can be physically separated by sorting. Unique flow cytometric capabilities in the resource include high-resolution chromosome analysis and sorting, fluorescence lifetime measurement, rapid-mix analyses with subsecond time resolution, phase-sensitive fluorescence detection, DNA fragment size quantification, ultrasensitive fluorescence detection, full spectral analysis, and a robust, flexible digital data display and analysis system. Expert advice and assistance are available to collaborators in the areas of cellular and chromosome sample preparation, multiplexed genetic and protein analysis, rapid kinetic analyses, macromolecular assembly dynamics, intrinsic property analysis, multivariate data acquisition and analysis, sorting procedures, and instrument development and troubleshooting.

Current Research

The resource has active research programs in the following areas: microsphere- and cell-based analysis of molecular interactions, kinetic analyses of signal transduction processes in cells, DNA fragment-size distribution analyses, analysis of isolated subcellular organelles, high-throughput screening of molecular interactions, full spectral flow cytometry, phase-sensitive flow cytometry, novel detection methods (photoacoustic and magnetic), miniature flow systems, and digital data acquisition and analysis. Associated research includes collaborations in cell cycle analysis and control, applications of chromosome analysis and sorting in neoplastic transformation, protease analysis, pathogen detection and identification, viral particle analysis, host-pathogen interactions, rapid genotyping, protein evolution, tumor biology, protein ligand development, and medical applications of flow cytometry.

Resource Capabilities

Methods

The resource has developed novel methods in several areas of flow cytometry, including protease activity assays, chromosome preparation for analysis and sorting, rapid single nucleotide polymorphism (SNP) analysis, DNA fragment size analysis, two hybrid protein interaction screening, analysis of individual mitochondria, cell-based protein binding assays, and multiplexed protein analysis assays.

Instruments

The resource's User Facility contains the following commercial instruments: two multiwavelength sorting systems with three lasers for sequential excitation (BD Vantage and BD Aria), a three-wavelength analyzer with a multiwell plate sample loader (BD LSRII), two five-parameter analyzers (BD FACSCaliber), a multiplexed bead analysis system (Luminex), fluorescence microscope with cooled charge-coupled device (CCD) camera, static and time-resolved spectrophotometers, and a scanning spectrofluorimeter. It also has seven custom instruments available for use by collaborators: phase-sensitive, fluorescence lifetime analyzer; rapid mix flow cytometer; dedicated DNA fragment-size analysis cytometer; a high-sensitivity cytometer; acoustic focusing cytometer; magnetic sensing cytometer; and a full spectral resolution analyzer.

Software

Open Reconfiguable Cytometry Analysis is a totally digital data acquisition and analysis system for collecting and processing flow cytometric data from a wide variety of analog (photomultiplier tubes, photodiodes, photoacuostic, and magnetic) and digital (CCDs and photon-counting) sensors. This modular system is currently running several of our custom instruments and is portable across a variety of computer platforms.

Special Features

The custom instruments in the resource have the ability to capture several types of unique information about cells and particles, including subsecond kinetics, full emission spectra, fluorescence lifetime, magnetic properties, and individual molecule analysis.

Available Resources

All of the commercial instruments in the User Facility are available to internal users and external collaborators, currently on a no-cost basis. The custom resource's instruments are generally available for use on collaborative projects, and >200 man-years of experience and expertise in flow cytometry instrument development and applications are available for collaboration and consultation.

Training Opportunities and Workshops

The resource, in collaboration with Verity Software House, sponsors an annual course in flow cytometry, featuring a distinguished international faculty and intensive hands-on laboratory training. In addition, the resource's personnel provide a traveling course entitled "Build Your Own Flow Cytometer," which teaches the basics of flow cytometry in an interactive, hands-on mode. The resource's personnel also participate as instructors in several other flow cytometry courses.

Publications

1. Goddard, G., Martin, J. C., Graves, S. W., and Kaduchak, G., Ultrasonic particle-concentration for sheathless focusing of particles for analysis in a flow cytometer. Cytometry 69A:66–74, 2006.

2. Ferris, M. M., Yoshida, T. M., Marrone, B. L., and Keller, R. A., Fingerprinting of single viral genomes. Analytical Biochemistry 337:278–288, 2005.

3. King, J. B., Gross, J., Lovly, C. M., Rohrs, H., Piwnica-Worms, H., and Townsend, R. R., Accurate Mass-Driven Analysis for the Characterization of Protein Phosphorylation. Study of the Human Chk2 Protein Kinase. Anal. Chem 78 (7), 2171–2181, 2006

4. Graves, S. W., Woods, T. A., Kim, H., and Nolan, J. P., Direct fluorescent staining and analysis of proteins on microspheres using CBQCA. Cytometry 65A:50–58, 2005.

5. Yan, X. M., Zhong, W. W., Tang, A. J., Schielke, E. G., Hang, W., and Nolan, J. P., Multiplexed flow cytometric immunoassay for influenza virus detection and differentiation. Analytical Chemistry 77:7673–7678, 2005.