Cell Sorting: An Enriching Experience

Click to view the PDF file: Suppliers of Flow Cytometers with Cell Sorting Capabilities Courtesy of CytomationCytomation's MoFlo MLS, capable of sorting 100,000 cells/second. Flow cytometers equipped with cell sorters enable the isolation of highly purified cell (or other particle) subpopulations. Unlike bulk separation methods, flow cytometry-based purification is on a "per particle" basis. Each particle is classified based on any of a number of different properties prior to sorting. In this article, The Scientist shines the spotlight on commercially available options for flow cytometers with sorting capabilities. Most cytometers are for analysis only, and are not equipped with sorters. Those that have sorters tend to fall into one of two very broad classes. Flow cytometers equipped with high-speed sorters are primarily used for "heavy duty" research applications. As a group, these instruments emphasize flexibility, making them adaptable for use in diverse research applications. They tend to have multiple, high-power lasers and are capable of simultaneously detecting and sorting based on multiple fluorescent parameters. Many offer modules for specialty applications, such as cloning or large particle sorting. In contrast, flow cytometers with low speed sorters primarily serve as workhorses in smaller-scale laboratories. These instruments are generally capable of detecting fewer fluorescent colors than their high-speed counterparts, but are less expensive to purchase and maintain. Flow Sorting 101 Today's flow cytometers use a sophisticated array of fluidics, optics, electronics, and computer software. Suspended cells are injected into a laminar flow stream and forced to flow, single-file, through a focused beam of light (usually laser). Light scatter and fluorescence emission signals are sensed, digitized, and displayed as frequency distributions. Light scatter alone is quite useful, providing a "morphological footprint" of sorts for each particle. Low angle forward scatter intensity yields information regarding cell size and viability. Side scatter tends to be more affected by inclusions within cells; it is a rough indicator of morphological features such as cellular granularity, membrane complexity, and organelle number. In most research applications, cell sub-populations are defined via the use of multiple fluorescent probes (e.g., monospecific antibody conjugates). Most clinical instruments employ a lower wattage, air-cooled, argon-ion laser that is preset to emit light at one wavelength (488 nm). The higher wattage lasers featured in most research-oriented instruments create more heat and require a more expensive water-cooled system. Multicolor analyses allow researchers to employ different fluors in defining subsets. Contemporary high-end instruments provide multiple lasers along with the beamsplitters, mirrors, filters, photomultiplier tubes, and signal-processing components required for simultaneously measuring signals from different fluors. Most employ a higher wattage argon-ion laser that can be tuned to different emission wavelengths (315 nm, 457 nm, 488 nm, or 515 nm). Other popular choices include helium-neon (633 nm) and krypton-ion (seven different wavelengths falling in the range of 350-647 nm). Dye lasers, in which laser beams are emitted through a liquid dye to yield a secondary beam of a different wavelength, are also in use. In flow-based sorting, particles are either isolated or discarded based on predetermined criteria (light scatter and/or fluorescent emission parameters). Sorters can be broadly classified as "mechanical" or electrostatic. Mechanical sorting systems are closed, preventing both contamination and evaporation; however, they are relatively slow. Most commercially available sorters employ electrostatic deflection, which enables much higher sorting speeds. These instruments are generally equipped with aerosol containment systems for user safety, as well as routines for sterile sorting. Historically, two basic types of flow chambers have been employed for sorting applications. Most high-speed, drop deflection-based sorters employ a "jet-in-air" method, in which the laser beam crosses the flow stream after it leaves the nozzle. Better optical data can be obtained if the sample is examined while in a cuvette ("sense-in-quartz"); this methodology has generally been used in conjunction with mechanical sorters, which have slower sorting rates. Elements from both worlds have been fused in a system from Beckman Coulter of Fullerton, Calif. Samples are probed while in a quartz cuvette, then released through an aperture for subsequent high-speed, drop deflection-based sorting. In electrostatic deflection-based sorting, the flow stream is broken into uniform-sized droplets via vibration of the flow tip (nozzle) at a precise frequency shortly after scanning by the light source. The time period from the examination of a particle to its inclusion in a newly formed droplet ("drop delay") is constant. Thus, droplets containing particles with the desired properties can be specifically charged upon formation. An electronic system confers the appropriate charge to each droplet after processing the light scatter and fluorescent signals from the particle within. The droplets then pass through an electromagnetic field established by two charged deflection plates. Each charged drop deflects towards the plate of opposite charge, and is collected in a tube, microplate, or other vessel. In most cases, only two populations are simultaneously isolated, because uncharged droplets fall into a waste container and are discarded. However, this limitation can be overcome by separating based on degree of charge. Different sorting protocols can be employed, depending on the application. For example, the duration of the charge pulse can be increased to include more than one droplet, to minimize the chance of losing sample owing to uncertainty about the drop count. In a common scenario, three droplets (the one believed to contain the targeted particle, along with the two flanking droplets) are collected. Many also choose to include a number of empty droplets (usually ten) between each particle. This decreases the chances of collecting unwanted particles; however, the particle flow rate, and hence time efficiency, is also decreased. A Tale of Two Sorters BD Biosciences of San Jose, Calif., offers two lines of modular, automated flow cytometers with sorting capabilities. FACSCalibur™ has a low-speed mechanical sorter, whereas the FACSVantage™ series of the company's top-line instruments features high-speed drop deflection sorters. The Sort Enhanced (SE) edition of the FACSVantage series is the company's high-end system, capable of sorting rates of up to 25,000 events per second. The FACSVantage SE employs up to three lasers and can be used for the detection of up to six fluorescent signals. This system is flexible in that the user can choose between a variety of different lasers and can reposition and exchange the lasers of choice. Its AccuDrop™ feature simplifies instrument setup for cell sorting operations by allowing automatic drop-delay calculations directly from the video monitor. The DiVa option provides a wide array of user-definable sort modes after automated drop-delay calculations have been made and stored in the instrument. Each mode enables the user to optimize for purity, recovery, or count accuracy. Nonrectangular sort gates can be employed, and up to 16 cell subsets can be identified using gates of various sizes and shapes. Optional modules for specialized applications include MacroSort™ plus, which enables sorts of larger cells, and CloneCyt™ Plus, for cloning applications. FACSCalibur is a dual-laser, benchtop system capable of detecting up to four colors. With sorting rates of up to only about 300 events per second, this instrument is primarily used in small academic research laboratories and clinical settings. An attractive feature of this instrument is that it does not require complex or time-consuming setup procedures. During sorting, the user simply gates the population of interest at the computer terminal, makes one click of the mouse, and the system does the rest. An optional module enabling automated sample injection from either tubes or microtiter trays is also available. The FACSCalibur and FACSVantage systems both employ the FACStation™ system and CELLQuest™ software for instrument setup and control, data collection, analysis, and management. BD Biosciences also offers a remote diagnostics service that aims to make troubleshooting easier and more efficient for the client as well as for the support specialists. The researcher dials directly into the company's flow cytometry support services from the computer terminal; BD staff members are able to see on their computer screen exactly what is seen on the client's terminal. On the Quartz Beckman Coulter's Epics® ALTRA™ flow cytometer can analyze and sort on up to six colors simultaneously. The ALTRA System was designed for the rapid separation of large numbers of specific cell populations. "One of the unique advantages of the EPICS ALTRA system is its ability to use small, air-cooled lasers while maintaining excellent sensitivity and resolution. This is due, in no small part, to Beckman Coulter's quartz flow cell design," comments Grant Howes, marketing manager of cytometry at Beckman Coulter. The base system is capable of sorting at rates of up to 10,000 cells per second (with small air-cooled lasers) or up to 15,000 cells per second (with water-cooled lasers). Those interested in even higher throughput might wish to consider the optional HyPerSort™ system, which uses high pressure and high frequency to increase sorting rates to up to about 30,000 cells per second. The HyPerSort system can be purchased along with the system or obtained later as a separate add-on module. The ALTRA system features eight different sort modes, four of which optimize between purity and yield for sorted populations, three of which feature the new ALTRASort™ mixed-mode option, and one that employs AccuSort™. Designed for working with rare or precious populations, the ALTRASort mixed-mode option isolates a high purity population to the left and captures the remainder of the same population to the right. The AccuSort mode is for accurate counting when sorting individual cells for cloning with the Autoclone® sorting option, or for sort matrix verification. The standard ALTRA can sort particles of up to approximately 70 µm. Beckman Coulter also offers the Large Particle Sorting (LPS) module, which provides three additional orifice sizes for the sorting of larger particles (up to 100 µm, 140 µm or 200 µm). The optional Forward Fluorescence Detector (FFD) improves measurement sensitivity of forward scatter. The FFD module provides some additional versatility to the system, enabling, for example, the analysis and sorting of submicron populations (e.g., bacteria or marine algae) or nonspherical cells (e.g., sperm). The ALTRA System includes the Coulter FlowCentre™ multi-media workstation, and employs EXPO32 Software for instrument control, data acquisition, and analysis in a Windows® 98 environment. In the Super-fast Lane Fort Collins, Col.-based Cytomation's MoFlo® Modular Flow Cytometers incorporate proprietary, patented technologies developed at Lawrence Livermore National Laboratory of Livermore, Calif. Designed for high speed, MoFlo systems sort up to 70,000 events per second in the purity mode, and up to 100,000 events per second in the enrichment mode. High sorting rates are particularly attractive to researchers seeking to isolate rare cell sub-populations. The company says that high cell viability and purity (greater than 99 percent) are achieved even at these breakneck speeds. MoFlo instruments accommodate up to three different lasers. The standard systems can monitor 14 fluorescent and two light-scatter signals. "Besides the 16 measured parameters, the electronics can calculate 16 additional parameters, for a total of 32," comments Sonja Wulff, spokesperson for Cytomation. MoFlo systems currently sport 15 sorting modes. A range of nozzle orifice sizes (50 µm, 70 µm, 100 µm, 150 µm, and 200 µm) enable purification of particles of different sizes. The Windows NT-based Summit™ software system is employed for instrument control and data collection/analyses. Currently, researchers can choose between three basic set-ups, which use the same core equipment but come with different modules. The MoFlo MLS (multi-laser sorter) is designed for the fast-paced research environment, and the MoFlo BTS (bench-top sorter) is for smaller-scale laboratories. The MoFlo HTS (high-throughput screening) features modules for the high-throughput environment, including an automated sampler, a microplate movement system, and an automated cloner for the single-cell deposition of cells into microplates. The company also offers a system specifically designed for separating sperm containing X-chromosomes from those bearing Y-chromosomes (MoFlo SX). Magnetic Attraction The primary limitation of flow cytometry/sorting is time efficiency. In previous years, most sorters were capable of working at rates of up to several thousand events per second. This has driven the development of the higher-speed sorters now on the market. However, considerable (even impracticably long) lengths of time might still be required for the isolation of rare subpopulations. There are finite limits to just how fast cells can be sorted using current sorting modalities; at some point, there is a tradeoff between increases in speed and decreases in recovery, purity, or viability. Courtesy of Miltenyi BiotecMiltenyi Biotec's autoMACS can be used to enrich for a cell population prior to sorting. Time efficiency can be increased via the partial purification of the cells of interest with a bulk separation method prior to flow sorting. A review of bulk sorting techniques would require its own article. However, Bergisch Gladbach, Germany-based Miltenyi Biotec's bench-top autoMACS (automated magnetic activated cell sorter) will be noted because it is sometimes found in cytometry facilities and is fully compatible with flow cytometry. Cells are first magnetically labeled with super-paramagnetic MACS MicroBeads that are coupled to an appropriate secondary reagent (e.g., anti-fluorochrome, anti-IgG, or streptavidin) and then passed over magnetic separation columns placed in a strong magnetic field. The magnetically labeled cells are retained in the column, while nonlabeled cells pass through. After removal of the column from the magnetic field, the magnetically retained cells are eluted. The MACS MicroBeads are biodegradable, and the company says that the beads do not activate cells, influence function, or affect viability. Further, the MACS MicroBeads are submicroscopic (approximately 50 nm in diameter) and do not affect the light scatter properties of labeled cells. Thus, after autoMACS sorting, the cells can be subjected to flow cytometric analysis or further flow cytometry-based sorting without prior removal of the magnetic spheres. The autoMACS enables the simultaneous sorting of multiple samples. The company also offers the CliniMACS for separating large numbers of cells in a closed, sterile system, as well as the miniMACS for small-scale applications. The ability to precisely sort cell populations based on the presence or absence of phenotypic markers has been a tremendous boon to immunology, cell and molecular biology, and related branches of the life sciences. It allows researchers to study more complex questions than was previously possible, by effectively amplifying the experimental signal-to-noise ratio. However, because cell sorters are so expensive (relative to flow cytometers), they are often found in core facilities. Thus, researchers are encouraged to first contact these centers if they are interested in harnessing the power these instruments offer. Deborah A. Fitzgerald (df@sciwriters.com) is a freelance writer in Birmingham, Ala. References 1.L. Bogh, T. Duling, "Flow cytometry instrumentation in research and clinical laboratories," Clinical Laboratory Science, 6:167-73, 1993. 2.A. Orfao, A. Ruiz-Arguelles, "General concepts about cell sorting techniques," Clinical Biochemistry, 29:5-9, 1996. 3.H.J. Tanke, M. van der Keur, "Selection of defined cell types by flow-cytometric sorting," Trends in Biotechnology, 11:55-62, 1993. 4.A. Givan, Flow Cytometry: First Principles, New York, Wiley-Liss, pages 15-73, 1992.

Cell Sorting: An Enriching Experience

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