Going with the Flow

In January Tim Bushnell, scientific and technical director of the University of Rochester Medical Center’s Flow Cytometry Core in New York State, packed a $50,000 flow cytometer in his car and drove it to a lab 15 minutes away. There, he trained beginners to use the technique—which identifies and sorts specific populations of cells—on the new benchtop machine. Just a few years ago such portability would have been unheard of: flow cytometry is generally done by teams of highly trained experts at core facilities, working on massive machines costing more than $100,000—in some cases $500,000. That is changing fast as a surge of new instrumentation is making flow cytometry easier and more affordable. Today’s systems can be built for benchtop use and are getting ever cheaper. Much of the machine setup is now automated, which will likely continue to democratize the method. A wealth of reagent kits is also taking the guesswork out of assay development and thus lowering the barriers for beginners. Large companies such as Sony and BD are investing in and marketing instruments costing under $100,000 and targeted to individual researchers rather than core facilities. But the new instruments haven’t made the art of flow completely foolproof, and users should understand what the technology can and can’t do before purchasing one of these systems. “Of the labs here who have bought their own instruments, some come back and say, ‘We kind of bit off more than we can chew,’ ” says Ryan Duggan, technical director of University of Chicago’s flow cytometry core facility. Personnel at core facilities are less familiar with the new machines, which may leave you relying on the manufacturer to troubleshoot. The Scientist surveys the options currently on the market and breaks down the pros and cons of some of the new machines. Accuri C6 Flow Cytometer Overview In March, BD acquired Ann Arbor, Michigan–based Accuri Cytometers and its flagship product, the Accuri C6, a two-laser, four-color instrument that has become popular with individual research labs—and even in some shared university facilities. Pros Accuri C6 eliminates a common pitfall for new users: adjusting the voltages to keep different cell populations on the same scale. Normally, if you’re off in your voltage settings, “there’s no real way to recover that data without rerunning the samples,” Duggan says. It’s simple to install and maintain, says Neal Benson, scientific director of flow cytometry at the University of Florida in Gainesville. In addition, the parts inside the instrument, like the peristaltic pumps, are fairly common, meaning that you can keep extras on hand and swap in and out as needed, Duggan says. “It’s not super sophisticated on the inside with tons of valves and switches,” he adds. If they can’t help you troubleshoot, Accuri’s support service will ship you a temporary replacement instrument while they fix yours. The instrument’s software package, called CFlow, is utilitarian and visually pleasing, consisting of the bare minimum you need to collect and analyze data—ideal for a lab that is going to do only a few applications, Duggan says. www.accuricytometers.com Cons The C6 software is still evolving and lacks some desirable features. For example, the software doesn’t produce publication-quality images because the graphics aren’t scalable, Bushnell says. He and others use third-party software, such as FlowJo, to help with analysis and data display. The machine is physically limited in its ability to accommodate more lasers and detectors. The C6 is slower to rinse out the system between samples than some other machines, so if you have many samples your runs could take a while, Benson notes. (Companies such as IntelliCyt make flow-cytometry samplers compatible with the C6 that can boost throughput.) Millipore Guava easyCyte family including the EasyCyte 5, 6 and 8 Overview These machines employ silica microcapillaries instead of sheath fluid, the liquid stream that carries cells through the detector in most traditional machines. The most basic version, EasyCyte 5, is a single-laser instrument that can be used to see three colors. Pros By eliminating sheath fluid users can run assays with a smaller sample volume. This simplifies the fluidics running through the system and eliminates the need to purchase or prepare sheath fluid—or troubleshoot it if you suspect it’s contaminated. Offers easily preset applications, such as apoptosis detection using markers like Annexin V. Standard assay kits come with their own software modules to help you interpret your data. Setup and maintenance is automated, meaning that inexperienced users can operate the instrument somewhat successfully. Cons Automation makes the machine a less flexible option for core facilities and more experienced users, says Duggan. In theory, capillary systems might be more prone to clogging compared with traditional flow cells, notes Benson. In particular, large particles could slow the flow, perhaps causing a buildup of following particles in the stream, he says. (Benson doesn’t know whether the Guava is prone to this problem in practice. “And, of course, it does happen in all flow cytometers from time to time,” he adds.) www.millipore.com Attune Acoustic Focusing Cytometer Overview Developed by scientists at Los Alamos National Laboratory and sold by Applied Biosystems (Life Technologies) starting last spring, this first-in-class flow cytometer uses ultrasound waves rather than fluid to align cells at the detection point. The Attune comes with two lasers and allows for analysis of up to six colors. This instrument was named one of the Top Ten Innovations of 2010 by The Scientist. Pros Acoustic focusing allows you to push more volume through the instrument in a shorter amount of time and still keep the cells aligned in the focal point. This is ideal for dilute samples or populations of rare cells such as stem cells, says Duggan. Conversely, acoustic focusing can be used to slow down the flow of cells if you want to boost sensitivity, notes Alan Saluk, director of the flow cytometry core at the Scripps Research Institute in La Jolla. The instrument includes a violet laser that is higher powered than the standard lasers used in other affordable cytometers; Life Technologies has optimized probes for the instrument. Cons The Attune does not come with a red laser, which is popular among flow users. For the more typical types of flow scenarios, where you run smaller (µL) volumes at a higher concentration, acoustic focusing might be overkill—and not worth the higher price tag, Duggan says. www.appliedbiosystems.com Is “AFFORDABLE” flow cytometry for you? List your goals. There are some things the new machines won’t do. For example, if you’re going to be looking at submicron-resolution particles, none of the low-end instruments will be optimal. For the fluorescence tests he runs on cytometers, Duggan determined that budget cytometers are also not as good at distinguishing dimly lit cell populations as the more expensive full-size models such as the BD FACSCanto II, BD LSR II, and Beckman Coulter Gallios. This might matter if, for example, you’re trying to parse groups of phosphorylated and unphosphorylated receptors. Ask yourself where you will be in five years. How many samples and what kind of assays will you be running? What parameters will you be looking at? Do you expect to increase the number or complexity of your applications, and is it possible to upgrade the machine you’re considering? If the instrument doesn’t meet your future needs, you might just stick with a core facility, Saluk says. Factor in ongoing expenses. You’ll be paying not only for assay reagent kits, filters, and other consumables, but also for replacement parts and the time it will take to install them. A Guava EasyCyte flow cell costs up to $400 and lasts about six months, if it’s well cared for. More traditional flow cells can cost thousands. And don’t forget service plans: Benson spent $3,700 on one for his Accuri C6 last year in addition to spending a few hundred dollars to maintain the instrument. Test it. If you do decide to take the leap, contact your core facility and ask them whether they’ve demo’d the instrument. Some core facility directors recruit users to run relatively common applications on demo machines, and report how the results compare to other instruments. Others, such as Duggan, run their own assays to assess performance on parameters they are most interested in. If you don’t have access to a core facility, ask for a demo in your lab to check how easy a model is to use and maintain. “If you’re paying $50K for an instrument and it’s down 50% of the time, you’ve wasted money,” Bushnell says. Educate yourself. Software that comes with the instruments won’t prevent you from accidentally misidentifying background fluorescence as a true signal. (Programs also won’t tell you which controls to run to prevent these mistakes.) “You need to be very careful—[data misinterpretation is] where I suspect the biggest danger is in most low-cost instruments,” says J. Paul Robinson, director of Purdue University’s Cytometry Laboratories in West Lafayette, Indiana. That’s why you should invest time in flow cytometry training. There are free introductory courses online and myriad books published on flow, some of which are specific to a field, such as stem cell biology. Better still, seek out experts. Many institutions offer free short courses, and there is an excellent paid course, the Annual Course in Flow Cytometry, held alternately at the University of New Mexico in Albuquerque or Bowdoin College in Brunswick, Maine, Saluk says. This year it will be held on June 11 at the University of New Mexico. Budget-friendly flow cytometers Instrument Price*(in US dollars) Size (inches) Lasers Emission detection Additional parameters Sample format Partec CyFlow Cube 6 www.partec.com approx. $35,000 approx. 14H 18.5W 20D 488 nm and 638 nm lasers are standard, but can be customized 4 colors, user-changeable filters Forward and side scatter; absolute cell counts Standard Eppendorf or Grainer sample tubes, 3.5 mL or similar; upgrade for more flexibility Millipore Guava EasyCyte 5 www.millipore.com approx. $45,000 8.75H 17.75W 17.25D 488 nm 3 colors Forward and side scatter; absolute cell counts 1.2 or 1.5 mL tubes Accuri C6 www.accuricytometers.com $49,000 11H 4.75W 16.5D 488 nm and 640 nm (upgrade to select laser configuration) 4 colors, user-changeable optical filters Forward and side scatter; absolute cell counts 1.5 mL tubes; upgrade to CSampler for more flexibility Stratedigm SE500 www.stratedigm.com $68,000 or less 24H 21.5W 21D 488 nm and 640 nm 4 colors, user-changeable filters Forward and side scatter 12 x 75 mm tubes or bullet tubes; upgrade to plate/tube loader SiCyt Eclipse www.i-cyt.com $70,000 (for single-laser machine) and up 21H 19.5W 24.5D 1 laser (488 nm); can add up to 3 more (405 nm, 561 nm, or 642 nm) 5 colors, user-configurable detectors Forward and side scatter; cell volume (“electronic volume measurement”); absolute cell counts 12 x 75 mm or 1.5mL tubes; assorted plates Attune Acoustic Focusing Cytometer www.appliedbiosystems.com Less than $100,000 16H 23W 17D 405 nm and 488 nm Up to 6 colors Forward and side scatter; absolute cell counts Accommodates 17 mm x 100 mm to 8.8 mm x 45 mm tubes * includes software and one-year warranty

Going with the Flow

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