by Caitlin Smith
“The applications for flow cytometry are so broad that flow cytometry has become an indispensable tool for cell biology research,” says Lew McAllan, general manager of global research cytometry at iCyt Mission Technology. Not long ago, you had to wait your turn to use a flow cytometer in a core facility—now you can have your own in your office. Today’s sophisticated technology has made cell analysis better than ever. If you are searching for a flow cytometry system, here are some considerations and examples of the features available today.
Purpose and ease of use
One of the first things to consider is what types of experiments will be performed with the flow cytometry system. In addition, consider the ease of use of the system, which often has a lot to do with the system’s software. Labs with many users may benefit from software that requires users to sign in, so that data can be easily correlated to each individual. Also, consider whether you may need biosafetyprotection. This is available, for example, in iCyt’s Synergy system, which offers the option of housing the system’s sorting components inside a certified, Class II Bio Safety Cabinet.
User-friendly systems that researchers are able to run with little or no prior training are helpful, but it is prudent to ensure that advanced functions also are available for experienced users. “Look for an instrument with a software package that enables you to get up and running in a short period of time, but make sure the software has expanded capabilities so that you are able to perform more complex analyses in the future,” says Jason Whalley, market manager for flow cytometry at EMD Millipore. “We train researchers to perform assays on our systems within a morning. However, as researchers become more comfortable and experienced with the instruments, our InCyte software is capable of more sophisticated experimental setups and analyses.” InCyte software from EMD Millipore simplifies data analysis using “a visual representation of target levels on a multiparameter basis via a heat-map display,” Whalley says. At the beginning of 2012, EMD Millipore will introduce a personal benchtop cellular analyzer for viability, apoptosis and cell cycle assays. The product’s patent-pending design enables its miniaturized flow cytometric technology within a toaster-sized instrument, and its MUSE™ system allows even inexperienced users to perform complex analyses. “The biggest challenge is complexity, which can limit access,” says Whalley. “Flow cytometry is still regarded as difficult to perform by inexperienced users, and this complexity needs to be simplified to broaden the adoption and utility of this powerful analytical technology.”
Many flow cytometry systems are housed in institutional core facilities, where they are used by many researchers from a variety of labs. These instruments must be especially user-friendly, yet also satisfy myriad research demands. “For a core facility, [one of] the key factors [is] ease of use, which mostly involves the software used to operate the instrument,” says Ryan Duggan, technical director of the Flow Cytometry Facility at the University of Chicago’s Cancer Research Center. “[Other important qualities are] flexibility, [or] the ability to change filters and optical configurations easily, and resolution, [as in] the ability to resolve dimly stained cells from unstained cells.”
Ease of use also means ensuring that a flow cytometry system will work well with other instruments in the lab. “For core labs, after quality and functionality comes compatibility with existing equipment,” says Paul Wallace, director of flow and image cytometry at Roswell Park Cancer Institute. “We train our users so they can properly use the equipment. They design their experiments around a particular platform and don't want to spend a lot of time learning new software programs or redesigning a protocol because we decided to change something.”
Flexibility, imaging and sorting
Another factor to consider is how flexible your system needs to be, especially if it will be used by many different groups. Customization can give you greater flexibility, as in EMD Millipore’s Guava easyCyte, part of a family of Guava systems that lets researchers customize their instruments. “They can be configured with one or two lasers, enabling detection of five to eight parameters,” says Whalley. “In addition, these instruments are available for different sample throughputs, from single-sample to 96-sample automated processing. All instruments use our proprietary micro-capillary technology.”
Another facet of flexibility is determining whether your flow cytometry requirements include imaging. EMD Millipore recently acquired Amnis Corporation, which adds imaging capabilities to fluorescent- detection flow cytometers like the ImageStreamX® and FlowSight®. “The ImageStreamX is the only instrument on the market today that combines the statistical power of flow cytometry with the insights of high-resolution microscopy of every cell,” says Whalley. “This enables researchers not only to obtain fluorescence data from millions of cells but also to visualize and quantitate the location of that fluorescence within the cell.”
The ImageStreamX imaging flow cytometer can give you up to 12 high-resolution images of each cell directly in flow, at rates exceeding 1,000 cells per second. “An imaging flow cytometer can do whatever a conventional flow cytometer does, but it also allows the researcher[s] to literally see the cells in their dot plots so they can detect artifacts as well as perform detailed quantitative analysis of hundreds of cellular parameters,” says Bob Smith-McCollum, marketing consultant at Amnis. “In addition to measuring simple fluorescence intensity, an imaging flow cytometer can measure the distribution of fluorescence intracellularly, quantitatively co-localize fluorescence signals and measure internalization.” The ImageStreamX was used by researchers at Children's Hospital Cincinnati to identify the dendritic cells responsible for Type I diabetes.
EMD Millipore/Amnis’ new FlowSight imaging flow cytometer is designed with a lower price (less than $100,000) for a broader market. “The FlowSight produces lower-resolution imagery than the ImageStreamX, but it is sufficiently detailed to discriminate single cells from doublets and debris, validate population gates and enhance cell cycle and apoptosis assays, all of which are strongly desired by flow cytometry users,” says Whalley. “The FlowSight can be further upgraded with a Quantitative Imaging option to enable a range of image-based applications.”
The flexibility to use a flow cytometer in new ways, for example in stem cell applications, also is valuable. Beckman Coulter’s MoFlo® Astrios™ combines new developments with features from previous instruments, such as the fluidic design of the original MoFlo and the processing capabilities of the MoFlo XDP. Pinholes separate each of the seven lasers for multi-color sorting, with laser alignment controlled by two stages for easier sorting. The intrument incorporates the IntelliSort II beadless drop-delay determination and monitoring system, and uses camera images and proprietary algorithms to determine the optimal drop delay. Beckman Coulter also offers a range of flow cytometry reagents, including CD markers (e.g. CD4, CD31, etc.) and a comprehensive selection of dyes that includes Krome Orange, a new violet-excited organic dye.
Recently, Beckman Coulter introduced a platform to automate stem cell differentiation. “The platforms integrate flow analysis and sorting instruments with automated plate handlers, dispensers, incubators and other required equipment for indicated processes to provide more consistent embryoid body formation and enhanced cardiomyocyte formation from murine embryonic stem cells,” says T. Vincent Shankey, principal staff advanced research scientist at Beckman Coulter Life Sciences. “Major improvements in the field of cytometry, particularly in the field of stem cell biology, will come from automated sample-prep systems, new reagents and new data-analysis tools to handle the increasing complexity of the data being generated, particularly by higher-end, multiparameter analyzers.”
The flexibility of a portable flow cytometer also can expand your horizons. Compact, portable benchtop systems such as the Guava are easy to use outside the traditional lab setting, expanding the number of flow cytometry applications. “We have thousands of Guava units installed with applications ranging from HIV monitoring in rural areas within sub-Saharan Africa, to analysis of cell viability in general-research and biopharmaceutical labs, to traditional analyses in immunology, cell signaling and stem cell research,” says Whalley. “We also have customers using our systems to identify the optimal algae strains for biofuels development.”
Another portable model is the BD Accuri® C6 flow cytometer from BD Biosciences. (Accuri Cytometers was recently acquired by BD Biosciences.) Customers find this system so easy to use that they are often, according to Grant Howe, Accuri’s vice president of marketing, “up and running out of the box,” with no training time. “The C6 has a miniaturized light path that makes it very stable, so no alignment is needed to set it up. It also uses fewer mirrors and filters, which means it loses less light,” saysHowe. The C6 has more than seven decades of dynamic range, so data is never lost—you simply use a zoom function to expand the area where the data is found. “Even a novice can use it,” says Howe, “because the wide dynamic range records ‘all the data, all the time.’” The C6 also uses peristaltic pumps combined with pulse dampeners for a stable flow, which means a smaller system with fewer moving parts and less maintenance. You can also make your own flow solutions for the C6 rather than purchasing them.
iCyt’s new iCyt® Eclipse™ Flow Cytometry Analyzer is another powerful benchtop model. “Its compact laser engine design provides future upgrade capability, as new laser modules can be added in the field,” says McAllan. “Depending on the available lasers, the user can run virtually any combination of fluorochromes or fluorescent proteins typically requiring research flow cytometers costing two to three times as much. The system allows easy optical filter changes to reconfigure the PMTs for different fluorochrome combinations.”
For sorting capabilities, iCyt’s Synergy™ Cell Sorting System can be configured with single or dual sorting heads. The dual configuration provides two independent cell sorters in one platform. “The Synergy can be configured for ultra high-speed cell sorting of up to 70,000 events per second or for routine cell sorting applications, both utilizing a new Quick Change precision-molded nozzle that replaces the conventional-style flow body and sort nozzle,” says McAllan. “This innovation in nozzle design is the result of a joint-development effort between Sony Life Electronics and iCyt and sets the stage for a true, affordable, disposable nozzle design in the future.” The Synergy system can be configured with up to seven lasers and 30 fluorescent detectors per channel, with the detectors located in the new Scalable PMT Array (SPA). “The SPA’s innovative design allows the user to easily configure any number of PMTs and detection filters for each laser collimation point, providing greater experiment flexibility for typical core lab applications [and] without the need to duplicate expensive PMTs,” says McAllan.
The most recent sorting flow cytometer release from BD Biosciences is the BD Influx™ Cell Sorting System, which uses up to seven lasers for two-way, four-way and six-way cell sorting. The flexibility in sorting configurations, along with a modular design, helps researchers tailor the flow cytometer to their specific applications. The new system also has different sorting modes, such as proportional and positional sorting, for a greater range of applications. Parallel electronics enable a throughput rate of 200,000 events per second. Yet even at high sorting speeds or high pressures, your cells are safe: BD’s special fluidics design uses an acoustic coupling in the sorting nozzle to create fluid droplets for the sorting process, meaning the cells experience a lower shear stress and stay healthier and happier.
Resolution and data quality
Although most flow cytometer systems offer high resolution and good data quality (this section is not intended to be exclusionary), there are several systems that offer unique routes to greater resolution that are mentioned here because they are perhaps less familiar to most readers. For example, Life Technologiesrecently released the Attune™ Acoustic Cytometer, the first acoustic focusing flow cytometer. Patricia Bresnahan, senior market development manager of flow cytometry at Life Technologies, says the instrument offers several advantages vs. most current systems. “The rate at which a sample can be analyzed is over 10 times faster than other analytical cytometers,” she says. “This allows researchers to process their samples faster and capture more data in a single day. Researchers looking for rare sub-populations of cells, such as tumor and stem cells, are particularly interested in this feature of the Attune Cytometer, because their experiments demand sampling millions of cells to accurately assess [the] 1% or less cells of interest. Secondly, the quality of the data is not compromised by this very rapid sampling rate, unlike other flow cytometers, where the data quality degrades as sample rate is increased. So for experiments demanding the highest quality of data, like cell cycle analysis and dim signal detection, this is a critical performance factor.”
Life Technologies soon will release an add-on plate runner/loader that lets you acquire samples not only from tubes but also from 96- and 384-well plates (in both shallow- and deep-well formats). Mike Olszowy, director of flow cytometry systems at Life Technologies, says functional reagents enhance the Attune’s advantages. “Most reagents are passive, identification-based reagents, such as antibodies for immunophenotyping, apoptosis or cell viability,” says Olszowy. “Life has built functional reagents like CellTrace™ Violet to trace the generation of cells as they divide, so now you can do GFP [green fluorescent protein] expression and cell proliferation or tracking/tracing at the same time.” Life also offers a new apoptosis dye that imitates annexin 5 staining, which shows a ratiometric change in fluorescence (called a membrane asymmetry probe) upon phosphotidylserine movement. “It reaches equilibrium in five minutes (compared to annexin, which takes about 45 minutes and requires special buffers),” says Olszowy. “Another advantage is that trypsinized cells tend to show false positives with annexin, but we don’t have that problem with our new dye.”
Stratedigm’s S1000 platform is designed for high resolution as well as flexibility. “All of Stratedigm’s flow cytometers are designed to provide unmatched sensitivity and resolution, enabling them to tackle demanding applications with dim signals such as micro-particle analysis,” says Rosa Chang, president of Stratedigm. The S1000 platform is designed for full scalability and full upgradeability after installation, so lasers and colors can be added later. “Stratedigm’s cytometers also come with [a] suite of unique patented and patent-pending technologies that allow for digital reassignment of detectors to spatially separated lasers, automated and remote diagnostics capabilities and Stratedigm’s analysis and acquisition software, CellCapture.”
Stratedigm recently upgraded the S1000 to the new S1000Ex flow cytometer, which uses the company’s proprietary Smart Detect™ design for the simultaneous detection of up to 14 colors. Stratedigm’s original S1000 cytometers can be upgraded to the Ex version. The Smart Detect technology uses one integrated PMT/electronics module to detect multiple fluorophores with similar emission wavelengths, but multiple excitation lasers. This new arrangement in optical detection may enable more researchers to use higher-performance, multi-color analyzers at lower prices than previously. Stratedigm is planning to launch a set of automation products for all of its analyzers in the near future.
Pre-enrichment of target cell populations also can aid in acquiring higher-quality data. Miltenyi Biotech offers this option in its MACSQuant Analyzer line of flow cytometers, which includes the MACSQuant Analyzer (violet, blue and red lasers, with nine parameters), the MACSQuant Analyzer 10 (violet, blue and red lasers, with 10 parameters) and the MACSQuant VYB (violet, yellow and blue lasers, with 10 parameters). These compact yet powerful cytometers can fit inside a laminar flow hood and are designed with automated features for ease of use. Features that have been automated for easy operation include start-up and shutdown, sample processing (tubes or 96-well plates), calibration and compensation. “What makes them truly unique is that they are the only flow cytometers on the market with an automated labeling of samples function and a magnetic pre-enrichment column,” says Shane Oram, global marketing manager for cell analysis at Miltenyi Biotech. “The automated labeling of samples function allows the MACSQuant to function as a robotic pipettor. It can add antibodies, buffers or dyes (such as propidium iodide or 7-AAD) directly to the samples before the samples are acquired.” The instrument’s magnetic pre-enrichment column, based on Miltenyi Biotech’s MACS technology, lets you pre-enrich rare cells before samples undergo flow cytometry analysis. “This offers a unique advantage to customers, as they can enrich their rare cells (such as circulating tumor cells or antigen-specific T cells) and then acquire thousands of events instead of millions of events to identify and analyze their cells of interest—saving time and maintaining small data-file sizes,” says Oram.
Large-particle flow cytometry
The ability to analyze large objects or clusters of cells in flow cytometers has opened up a new set of questions. “Cells in clusters communicate and behave differently than cells growing as a monolayer on the surface of a plastic dish or individually in liquid suspension,” says Rock Pulak, scientific director of Union Biometrica. “It is becoming clear that traditional cell culture approaches would benefit from considering three-dimensional culture techniques (cell clusters) where normal cell-cell interactions can be replicated. Additionally, using microcarriers made of (or incorporating) molecules of the extracellular matrix can more closely mimic the growth conditions normally found in the intact organism. Large cell clusters, including cardiomyocyte embryoid bodies, pancreatic islets and neurospheres, ranging from 50 [to] 700 µm, have been analyzed and dispensed based on their size, optical density and fluorescence intensity. Sorted cell clusters remain viable and functional after dispensing.”
Researchers who use common lab cell lines have many models of flow cytometers from which to choose, but those who need to sort larger cells or objects have basically two choices: sort them manually or use Union Biometrica’s COPAS Large Particle Flow Cytometers, which are designed to sort and dispense objects that are too big or fragile for traditional flow cytometers, according to David Strack, Union Biometrica’s chief executive officer and president. The four COPAS models are optimized for differently sized samples with flow cells of 250, 500, 1,000 or 2,000 microns. Types of samples that might be used in the COPAS system include large cells or cell clusters such as adipocytes, duct cells, hepatocytes, pancreatic islets or embryoid bodies; cells or combinatorial peptide libraries on beads in bead-based assays; embryos and larvae of model organisms such as Zebrafish, Drosophila or C. elegans; seeds like Arabidopsis; various types of algae; or organisms like Aspergillus.
“All Union Biometrica’s instruments are very gentle on the cells and organisms [being] analyzed or sorted, with little or no effect on viability,” says Strack. “Each offers our patented air-stream sorting mechanism that provides a gentle method for dispensing the desired subpopulation of cells or organisms for further study. Additionally, all Union Biometrica instruments work at low pressures, so samples experience only minimal shear forces that are insufficient to damage the cells and organisms under study.” The company also offers a new BioSorter that allows a full 10- to 1,500-µm range in a single system (which is particularly useful for shared instruments) and a high-throughput package for large screening applications.
The opportunity to work with larger objects in flow cytometry also provides the chance to collect positional information along the length of the cell cluster or organism as “it passes through the focused laser(s) and is interrogated for optical density and fluorescence,” says Pulak. “Profiler II records up to 8,000 data points per channel for each object and generates up to 21 measurements for analysis. This can be displayed as a profile or map of each fluorescence channel as well as extinction/optical density. These plots indicate the location of each fluorophore within the animal and allow detection of weak signals even in the presence of stronger ones.”
Finally, no matter what bells and whistles your flow cytometry system might sport, it always pays to consider the basics that will make life with your new instrument easier. These may include house-cleaning functions such as automatic start-up, shutdown or cleaning; partially or fully automated processing; and the degree and quality of customer support provided by the manufacturer. Keeping these criteria in mind will help you find the system that’s right for you.
Please note: This article was originally published on Nov. 22, 2010 and updated on Dec. 5, 2011.
The image at the top of this page is from the Amnis FlowSight.