Flow cytometry is a method for quantitating components or structural features of cells primarily by optical means. Although it makes measurements on one cell at a time, it can process thousands of cells in a few seconds. Since different cell types can be distinquished by quantitating structural features, flow cytometry can be used to count cells of different types in a mixture.
Flow cytometers have been commercially available since the early 1970's, and their use has been increasing since then. The most numerous flow cytometers are those used for complete blood cell counts in clinical laboratories -- these do not employ fluorescence. More versatile research instruments employ fluorescence, hence may be distinguished as flow cytofluorometers. Flow cytometric data can be seen in any issue of a scientific journal concerning cell biology. A large percentage of the papers in the American Association of Immunologists' Journal of Immunology report flow cytometric data.
Flow cytofluorometers are found in all major biological research institutions. They are also numerous in medical centers, where they are used for diagnosis as well as research. There are about 7,000 flow cytofluorometers in use worldwide. Ploidy and cell cycle analysis of cancers is the major diagnostic use. Lymphomas and leukemias are intensively studied for surface markers of diagnostic and prognostic value. Although less expensive alternative technologies are under development, until the present time, flow cytometry has been the method of choice for monitoring CD4 lymphocyte levels in the blood of AIDS patients.
In western Massachusetts, flow cytometry facilities are located at UMass Amherst, Amherst College, Baystate Medical Center in Springfield, and the UMass Medical Center in Worcester.
The term "FACS" is Becton-Dickinson's registered trademark and is an acronym for Fluorescence-Activated Cell Sorter. Another major vendor of flow cytometers is Coulter Electronics. Amherst College owns a Coulter Profile analyzer.
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How Does It Work?
The cells may be alive or fixed at the time of measurement, but must be in monodisperse (single cell) suspension. They are passed single-file through a laser beam by continuous flow of a fine stream of the suspension. Each cell scatters some of the laser light, and also emits fluorescent light excited by the laser. The cytometer typically measures several parameters simultaneously for each cell:
Light scatter alone is often quite useful. It is commonly used to exclude dead cells, cell aggregates, and cell debris from the fluorescence data. It is sufficient to distinguish lymphocytes from monocytes from granulocytes in blood leukocyte samples. Light scatter has been used in our Facility to quantitate aggregation of living cells.
Fluorescence intensities are typically measured at several different wavelengths simultaneously for each cell. Fluorescent probes are used to report the quantities of specific components of the cells. Fluorescent antibodies are often used to report the densities of specific surface receptors, and thus to distinguish subpopulations of differentiated cell types, including cells expressing a transgene. By making them fluorescent, the binding of viruses or hormones to surface receptors can be measured. Intracellular components can also be reported by fluorescent probes, including total DNA/cell (allowing cell cycle analysis), newly synthesized DNA, specific nucleotide sequences in DNA or mRNA, filamentous actin, and any structure for which an antibody is available. Flow cytometry can also monitor rapid changes in intracellular free calcium, membrane potential, pH, or free fatty acids.
Flow cytometers involve sophisticated fluidics, laser optics, electronic detectors, analog to digital converters, and computers. The optics deliver laser light focused to a beam a few cell diameters across. The fluidics hydrodynamically focus the cell stream to an within an uncertainty of a small fraction of a cell diameter, and, in sorters, break the stream into uniform-sized droplets to separate individual cells. The electronics quantitate the faint flashes of scattered and fluorescent light, and, under computer control, electrically charge droplets containing cells of interest so that they can be deflected into a separate test tube or culture wells. The computer records data for thousands of cells per sample, and displays the data graphically.
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Analysis Equipment: the FACScan
Our Facility provides a Becton-Dickinson FACScan for analysis of cell samples. Unlike the sorter (see below), this instrument cannot separate cells into different containers based on their properties; the samples are consumed and discarded during analysis. The FACScan is a closed fluidic system, so use with biohazardous samples (such as human blood samples) is possible with appropriate precautions and authorization.
The FACScan is easy to use, and in our Facility, the instrument is operated by the experimenter her/himself. See the separate section on training. The Facility maintains a phone-in reservation system so blocks of time can be reserved in advance for acquisition or data analysis.
The FACScan uses an air-cooled argon gas laser, 15 mW output, with a fixed wavelength emission of 488 nm. It has three fluorescence detection channels which simultaneously detect green, yellow-orange, and red light. Fluorescein is used extensively for the green channel, and phycoerythrin or propidium iodide (a DNA stain) for the yellow-orange channel. Dyes are also available which can be excited at 488 nm yet emit in the red.
The FACScan can analyze cell suspensions at the rate of several hundred cells per second. Typically, investigators acquire 5,000 to 15,000 cells per sample. Data are saved to the hard disk of a dedicated Hewlett-Packard computer, where they can later be analyzed with graphics software. Data can also be transferred to a network server computer so that they are accessible from any computer on the campus network. Excellent public domain software is available for PC's (not yet for MacIntoshes) which may be copied freely to any computer for data analysis.
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Sorting Equipment: the FACStar Plus
The Facility operates a Becton-Dickinson FACStar Plus for cell sorting, and for analysis requirements which cannot be met on the FACScan. This instrument has a class IV water-cooled argon gas laser with a rated output in all-wavelength mode of 4 watts. This laser can be tuned, so the FACStar is used, for example, when the analysis requires excitation at 514 nm in addition to 488 nm.
The FACStar Plus can sort cells, or acquire data, at a rate of several thousand per second (about 10-fold faster than the FACScan analyzer). Because the FACStar uses a stream-in-air sorting method, it aerosolizes the sample, and cannot be used for biohazardous samples. Nonhazardous living cells can be sorted, and may be recovered in gnotobiotic ("sterile") form for subsequent in vitro functional studies.
Unlike the FACScan, which is operated by the experimenter, FACStar Plus operation is restricted to trained operators (currently Cheryl Cote and Eric Martz). This is because the instrument requires much more training and experience in order for the operator to achieve proper alignment and function. Because of its open design, which permits great flexibility in configuration, its delicate components are much more easily damaged by inexperienced operators, and quite expensive to replace.
A recent use of the FACStar was to select a high-producing line from a hybridoma population. The hybridoma was producing monoclonal antibody at a very low level. Following a method recently reported in the literature, the few percent of cells with the highest surface immunoglobulin expression were sorted, and the resulting cell line produced antibody at a rate about 60-fold higher than did the starting population.
The FACStar is currently being used by Prof. Cynthia Baldwin and her students to purify bovine gamma/delta T lymphocytes for functional studies.
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Cost and Availability
New users are given several hours of free acquisition on the FACScan during their training period. New pilot projects may also be given a few hours of free time to establish feasability. Subsequently, fees are charged which partially defray instrument maintenance costs.
Investigators who are members of the Graduate Program in Molecular and Cell Biology (and hence who contribute a portion of the overhead from their research grants to the Program) are charged $40/hour for data acquisition on the FACScan. Use of the FACScan computer for analysis of previously acquired data is free. At present, there is ample time available on the instrument for non-member use. Non-members from the 5-college community are charged $50/hour. Rates for users from for-profit organizations are negotiated on an individual basis (please inquire).
Experienced FACScan users can acquire data at a rate approaching one sample per minute. Thus, the cost of use for MCB members can be slightly less than $1 per sample.
Operation of the FACStar sorter is restricted to properly trained personnel, presently Cheryl Cote or Eric Martz. If you are interested in sorting, please inquire about costs from one of these individuals.
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Location, Contacts, Phone Numbers
The UMass/Amherst Flow Cytometry Facility is located in Morrill I, room 428, as part of the Core Instrument Facility of the Graduate Program in Molecular and Cell Biology and the Program in Biotechnology. A packet of introductory training documents can be obtained from Cheryl Cote, the manager of the Core Instrument Facility, Morrill I room 426, 413-545-2485, ccote@mcb.umass.edu.
The faculty director of the Flow Cytometry Facility is Eric Martz, emartz@microbio.umass.edu, 413-545-2325. He is available for consultation regarding new projects, training, data analysis, and methods. The Facility subscribes to the journal Cytometry (since March, 1989, volume 10 number 2), and has a collection of methods books (contact Ms. Cote).
Reservations for FACScan use are taken by the MCB Office, 413-545-3246. The phone in the FACS room is 413-545-1933.
