2016 Flow Cytometry Buyer’s Guide: A Researcher's Guide to Selecting a Flow Cytometer

Flow cytometry has emerged as a powerful methodology to analyze single particles—most often cells from complex solutions. Innovations in instrumentation and supporting reagents have brought the technology to researchers en masse, including those who are exploring regulatory events both intra- and extracellularly. Flow cytometry enables researchers to more efficiently examine and characterize key markers such as small molecules, nucleic acids, as well as peptide and protein targets at the single cell level within the same experiment. Moreover, these analysis can be measured over time to better understand the regulatory mechanisms involved in cellular signaling, especially rare events.

Scientists have many instrumentation and reagent options when selecting and/or purchasing a flow cytometer system to address their research needs. The focus of this buyer’s guide is to discuss some of the key components and features of the instruments and to highlight some tips and considerations for selecting a flow cytometer system for your lab.

Background

The first commercially available flow cytometer instrument was launched in 1974. And for the better part of four decades, this laboratory workhorse has continued to evolve, helping scientists find ever smaller needles in the ever larger haystack of questions about the subtleties of how cells function. A technology that was once challenged to simply identify cells by size is utilized today to assist researchers in their immunotherapy studies and being used to diagnose diseases like cancer.

And yet, for all the innovations in fluidics, optics, labeling chemistries and analytics, the fundamental purpose of flow cytometry remains unchanged: the detection, quantitation and characterization of large biological particles.

Technology fundamentals

Tool providers offer many types of flow cytometry systems, primarily benchtop instruments as well as the growing focus on portable units that can be easily transported around, or even out of, the lab. Options and upgrades available on the different systems are tailored to address generic, exploratory discovery cell analysis experiments as well as extremely customized separations and cell or particle sorting.

When selecting a flow system, the three common components found in all systems are: fluidics, optics/detection and electronics/ informatics. Here we review the basic features of each of these components in more detail to provide assistance in selecting the appropriate options on your next flow system.

Fluidics

Flow cytometers take a solution of cells (e.g., blood, suspended culture or digested tissues) or particles and focus that solution into a stream that aligns the composition for downstream interrogation. The most common mechanism to focus this stream is hydrodynamic focusing, in which a nozzle ejects the cell solution through a stream of sheath fluid. 

The sheath fluid creates a drag effect around the cell suspension, essentially thinning the solution to the point that the suspended cells align single-file.

By adjusting the speed of the sheath fluid, you can adjust the diameter and therefore the throughput of the cell suspension; a faster sheath fluid means a narrower stream, increasing the likelihood that cells will move through the detector one at a time. This is useful, for example, in high-throughput drug screens or T-cell immunophenotyping.

When analyzing larger volumes or higher densities of cells, using a narrower stream tends to slow throughput, which can translate into a delayed time to results. Slowing the sheath fluid widens the stream and thereby increases throughput. But this improvement comes with an increased likelihood of multiple cells traveling side by side, convoluting the analytical signal, or the possibility of different cells passing through different parts of the detection beam, artifactually lowering the signals of some cells. In developing protocols, optimization of the appropriate flow rate vs. the cells or particles being sorted should be taken into consideration.

Acoustic focusing, using ultrasonic radiation pressure to move cells to the center of a stream, is an established alternative to either replace or augment hydrodynamic focusing. Acoustic focusing helps ensure that cells continue to pass through the system one at a time while allowing end users to maintain higher fluid throughput. Such an approach is particularly helpful when looking for rare events, such as circulating tumor cells in blood, when you may need to process large volumes to identify and characterize only a handful of cells. Acoustic focusing technology is utilized in Thermo Fisher Scientific’s Attune NxT instrument.

Recently, several tool providers, including MilliporeSigma (MUSE and Guava easCyte systems), Miltenyi Biotec (MACSQuant Tyto) and Cell Signaling Technology (CellSimple), have created flow instruments that eliminate the use of traditional sheath fluid by utilizing microfluids (often disposable) to focus the cell suspensions. Although not as elaborate in terms of features, options and potential levels of data reporting as higher-end flow systems, these alternative units provide powerful cell or particle analysis in a simplified and efficient package. For researchers with limited experience running flow experiments, or those monitoring their experimental expenses, these systems represent an attractive alternative that does not compromise results.

Optics

Upon focusing into a narrow stream, the single-file cells are interrogated by a light source—typically a laser.

The disruption and scatter of the light—defined as an event—provides information on physical properties of the cells.

In addition, some instruments contain multiple laser-light sources to enable the user to gain exponentially more data.

There are two measurements of light scatter that can be monitored in many flow systems. Forward-scattered light (FSC) offers insights on the size or cell surface area of the particles: Smaller cells refract less light, and larger cells refract more light. This is the more common measurement seen in all flow cytometry units.

The second measurement is side scatter light (SSC), which can provide information on the complexity of the cell—proportional to surface granularity or detection of internal structure. By combining these two measurements of light scatter, researchers can examine multiple parameters and features to better characterize their sample preparations. Monitoring the two types of light scatter enables researchers to identify and potentially differentiate cell types in complex, heterogeneous populations of cells.

Light-scatter patterns only provide physical descriptions of the cells. To interrogate the molecular characteristics of cells within a stream (e.g., gene expression, protein localization or cell-cycle status), the cells must be tagged in some way. Most often, this is done with fluorescent dyes or fluorescently tagged antibodies or nanoparticles.

Recently, polymer dyes (e.g., BD Biosciences’ Horizon and Sirigen reagents)—fluorescent markers that are several magnitudes brighter than conventional dyes—have helped researchers obtain finer levels of detection, particularly in the case of cell types and rarer events. Likewise, an expanding array of amine-reactive dyes is improving the quality control capabilities of flow cytometry and cell sorting by helping researchers identify and exclude dead cells from their collections.

Nanoparticles (e.g., Thermo Fisher’s Qdot Nanocrystals), meanwhile, can offer more consistent fluorescence for longer-term experiments, as they do not photobleach under constant illumination and can be monitored even when in vitro experiments become in vivo experiments, such as with the implantation of stem cells into animals.

The choice between dye, antibody and nanoparticle is largely dependent on the application and target. When targeting a very specific cell type—for example, CD4+ T-cells—a fluorescently tagged antibody can enable researchers to precisely quantify those cells within a mixture. But many applications (e.g., cell viability or cell cycle) do not require such molecular precision. In those cases, dyes may be more than sufficient.

Visit Biocompare’s product directory to view the selection of flow dyes from an array of vendors and review some of the latest options for dye selection that are not discussed in this article.

Because different fluorophores excite and emit at different wavelengths, most flow cytometers offer multiple light sources (lasers) and emission filters. Lasers used in flow instrumentation are typically: blue (488 nm), yellow-green (561 nm), red (640 nm) violet (405 nm) and ultraviolet (355 nm). Many tool-providers offer a variety of emission filters to use in combination with the different light sources his enables researchers to multiplex their experiments and monitor a variety of markers, whether different cell surface proteins, intracellular cytokines or shifting DNA content, reflecting the fluid nature of cell biology. A challenge in these experiments is optimizing the fluorescent tags and available lasers to minimize spectral overlap—the contribution of the emission spectrum of one fluorophore to the spectrum of a second fluorophore.

To some extent, this overlap can be mitigated by spectral filters that only allow the transmission of emission spectra either within a wavelength range (band pass filters), or above (long pass) or below (short pass) a specific wavelength. Alternatively, the newer fluorophores tend to offer narrower emission ranges, and instrument-specific software or online tools and freeware can provide guidance on how to select optimally distinct fluorophores.

Whether scattered or fluorescent, the light signal from the cell is collected by photomultiplier tubes (PMTs) that convert the light’s intensity into an electronic readout.

The sensitivity of the PMTs determines to some extent the resolution of detection—how small a particle or subcellular component you can detect, as well as how well you can distinguish signals from cells that pass close together.

In some instruments, particularly those that also provide image data, the PMT has been replaced with a CCD (charge-coupled device) camera. These instruments provide insights not only into the identity and intensity of fluorescent markers within a cell population but also into the precise locations of these markers within the cells. This can be helpful in experiments monitoring, for example, protein translocation and cell signaling.

The increased information generated by imaging-based cytometry, will generate increased file sizes which can result in potential data storage issues, but this is alleviated by pushing the data to cloud-based storage. In addition, specialized software to analyze and process the data is improving and becoming more user-friendly for those researchers with limited software analysis experience.

Table 1: Flow cytometer features overview

* FSC = forward scatter; SSC = side scatter;FL= fluorescence, N/A = not applicable.

Editor’s note, this table a brief overview of flow cytometry instruments and not meant to be an inclusive list of all commercially available instruments. Visit Biocompare’s product directory to view more detailed information on additional instruments, accompanying reagents and more information on their various features and applications.

Sorting

Identifying particles or cells is the first step in their characterization. Often further analysis is required, using techniques like next-generation sequencing (NGS) or digital PCR (dPCR), or it may be necessary to culture the cells for cell-based therapies. This is when flow cytometry transitions to cell sorting; researchers should consider their downstream workflow when selecting an instrument that has the ability to sort a subpopulation of the sample.

As the cell stream leaves the detection beam, a transducer causes the stream to vibrate, breaking it into single-cell droplets. Typical flow cytometers can produce 10,000 to 100,000 droplets per second.

The exact distance between event detection and droplet formation is called the drop delay. This is a critical parameter in ensuring that the droplet collected carries the cell that triggered the event, and the determination of drop delay has historically been a labor-intensive process requiring years of training. Recently, however, this process has been automated in several instruments (e.g., Bio-Rad S3e and BD Biosciences’ FACSAria Fusion) using internal optics and software, lessening the expertise required to set up the system.

Electrostatic sorting, in which a charge is applied to droplets of cells as they pass through an electric field, is one way to sort cells.

Embodying user-defined parameters, charged cells are deflected into a tube, plate or some other type of collection container. The uncharged stream, meanwhile, passes into the waste.

Different flow cytometers have unique methods to capture and sort cells from sample populations, so the best instrument for your lab really depends on sample type and the further characterization you want to perform. Multiple vendors offer flow systems that can sort cells. (See Table 1 for a few examples of instruments that offer this option).

User interface and analytics

Ultimately, most end users of flow cytometry systems see the technology as just one of many they require to achieve their scientific goals. Unless you are purchasing a unit as part of a core lab or expect large-scale use of the instrument, you may not want to dedicate a technician solely to flow cytometer use and maintenance. Rather, you may prefer a unit that requires minimal training.

To address researchers’ needs, tool providers have developed assay kits for numerous flow applications, ranging from detection of specific cell surface markers to detailed intracellular characterization assays. Biocompare lists a wide selection of assay kits for flow applications.

In addition, laborious and time-consuming processes such as alignment of lasers, sample streaming and even cleaning protocols have been automated on flow cytometry systems, to assist researchers. For example, the highly precise and time-consuming alignment of lasers and sample streams has been simplified, either through continuous, automated fine-tuning (e.g., Bio-Rad’s S3e AutoGimbal^TMsystem) or by fixing the optics in place to remove the need for adjustment (e.g., Thermo Fisher’s Attune NxT). And several instruments offer self-cleaning protocols and clog-detection systems designed to minimize unit downtime.

Likewise, experimental design and performance can be as simple as pushing a button, with instruments that offer standardized protocols and reagents that are approaching press, play and walk away. Such systems might be ideal for labs that expect to perform the same tests over and over again with very similar samples. For situations in which needs are more varied and less routine, however, software packages and user interfaces increasingly are being designed for a broader group of researchers—those who may not have expertise in designing flow experiments.

Similarly, data-analysis tools included with most instrumentation; software for purchase, such as FLOWJO; and public freeware are becoming simpler and more accommodating of distributed use throughout a facility. Most software packages let the users adjust results between display modes (e.g., histograms to dot blots), to isolate specific cell subsets (gating), to shift analysis parameters (e.g., from forward scatter vs. side scatter to forward scatter vs. fluorescence) or to generate heat maps of multiwell plates.

Applications

The number of applications that can be run on flow cytometers continues to grow. In many ways, the variety of applications to which flow cytometry is best suited is determined by the ability of the end user to identify a way to tag the cell. If you have a known target and can label that target, you can use flow cytometry and cell sorting to characterize and isolate cells carrying that target.

For these applications, many instrument and reagent manufacturers have standardized protocols and provide kits to simplify workflows. Although reagent manufacturers are keeping options open for researchers who choose or require to customize capture and detection reagents tailored to their specific target. While the focus is primarily on basic research discovery, scientists are expanding the utility of these applications as they gain a better understanding of how cells behave, and how they are regulated or impacted by both internal and external stimuli.

Here we highlight a few example applications that are commonly run on a flow cytometer. We also suggest visting the Biocompare product directory to view a listing of flow assay kits and reagents.

Immunophenotyping

This commonly run application is used to identify and quantitate populations of specific cell types based on the presence or absence of key biomarkers, such as surface proteins. The glycoproteins CD4+ and CD8+ are well-characterized surface proteins often used by researchers as a control marker when studying the immune system and various cell types. This research has expanded to hospitals and even field operations, where diagnosticians use fluorescently tagged anti-CD4 antibodies to examine levels of CD4+ T-cells in patients with HIV and monitor disease progression (dropping CD4 levels) or response to treatment (rising CD4 levels).

Cell-cycle analysis

Another common application for flow cytometry is the analysis of cells transitioning through cell division. Such assays are particularly useful for screening cytostatic drugs or examining ploidy differences in oncology specimens—much in the way cells would be studied by microscopy but with much higher throughput, a greater sense of cell-to-cell variability and the ability to detect multiple targets on a single cell.

Although early efforts at cell-cycle analysis relied on DNA dyes that were cytotoxic, a newer generation of dyes is allowing researchers not only to monitor cells by flow cytometry but also to continue analyzing those cells downstream.

Cell death/apoptosis

As with cell-cycle analysis, the ability to monitor cell mortality, whether through necrosis or apoptosis, can be vital in screening cytotoxic drugs or understanding the potential safety or toxicity of environmental and chemical agents. Alternatively, dysregulation of cell death can be a hallmark of disease, offering diagnostic opportunities.

These mechanisms of cellular destruction involve cascades of morphological and molecular changes within the cells—for example, release of microparticles, induction of caspases or altered mitochondrial membrane potential—that can be monitored by flow cytometry, down to single-cell precision.

Cell signaling

Flow cytometry may have started as a way to analyze extracellular markers, but improvements in molecular tagging and signal sensitivity have expanded the method to intracellular markers, such that signaling cascades and metabolic pathways can now be studied within a population of cells. But unlike methods such as liquid chromatography, 2D gel electrophoresis and Western blotting, which can only provide an overall picture of molecular changes, flow cytometry offers insights at the single-cell level. Combined with improved methods of fixation as well as more specific cell-permeable dyes, detection and characterization of intracellular activity have vastly improved.

Thus, researchers can determine which stem cells have achieved pluripotency, and which have started to differentiate. They can monitor protein phosphorylation events (e.g., BD Biosciences’ PhosFlow) following hormonal stimulation, or changes in cellular calcium flux (e.g., Fluo-4 AM, from BD Biosciences as well as other vendors) in a drug-screening experiment. A wide selection of flow dyes from a variety of vendors can be searched in the Biocompare product directory.

Cell proliferation

Just as unchecked cell proliferation is a hallmark of diseases such as cancer, diminishing proliferation rates can be a sign that a cell culture is struggling; it is not uncommon for slowing growth to be associated with increased cell line passage. In typical experiments, a culture is treated with dye (e.g., Thermo Fisher’s CellTrace) and then monitored over time. As the cells divide into daughter cells, the dye is distributed across increasing numbers of cells, and its signal diminishes. The rate of signal decrease suggests how quickly the cells are proliferating.

This information can be useful, for example, in identifying conditions that optimally maintain stem cells or in screening potential compounds that induce cell-cycle arrest. As well, by multiplexing with other markers, researchers can characterize and sort subpopulations of cells that may have useful or interesting characteristics, aside from a desired proliferation behavior.

Cell morphology (imaging)

Total fluorescence doesn’t necessarily tell the whole story of what is happening within a cell. Rather, the intracellular localization of a fluorescent signal, or morphological changes within subpopulations of cells, can provide valuable information about changes in cellular characteristics and behaviors. Imaging flow cytometers (e.g., IntelliCyt’s iQue Screener, MilliporeSigma’s ImagestreamX Mark II and Amnis FlowSight) combine the throughput of flow cytometry with the high-content imaging capabilities of microscopy, offering researchers a broader understanding of cell behavior. For example, researchers can monitor the ability of a patient’s cells to repair DNA damage, providing insight into how well a patient may respond to radiation therapy.

Many imaging experiments, however, do not require flow capabilities. High-content drug screens or simple cell counts, for example, can be performed using static platforms, such as slides or multiwell plates. For these applications, an image cytometer (e.g., Molecular Devices’ SpectraMax MiniMax, ChemoMetec’s NucleoCounter NC-3000 and GE Healthcare Life Sciences’ Cytell) may be sufficient, offering submicron resolution at multiple wavelengths for a variety of cell types.

Purchasing considerations

As discussed earlier, the basic components of a flow cytometer (or slight variations on them) are found in all systems. Determining which features of a particular instrument to select often depends upon the applications that are being run today, as well as what will be run in the near future. Based on a survey performed by Biocompare, here are a few tips to help you select the appropriate flow system for your research needs.

• Functional needs. Are you characterizing your cells, or will you require sample sorting? There are instruments that can perform one or both functions. And there are greater levels of separation, characterization and detection that can be achieved by the different systems or with upgradeable options.
• Identify users. Who will be using the system, and what is their level of expertise? Instrument, reagent and software tool providers have taken into consideration the diverse levels of expertise of users who may be using their technologies, and they have developed products to address the needs of all experience levels.
• Multiplex samples. The requirement to multiplex sample analysis will influence the number of lasers and the detection systems selected. Also, take some time to understand if the appropriate reagents (e.g., dyes and antibodies) are available to examine your target of interest. Biocompare lists a wide selection of antibodies and dyes for flow cytometry applications. Free online sources such as Chromocyte also provide guidance on antibody selection options. Often the samples being analyzed are very limited in quantity or a very precious material; multiplexing will generate more results, and it may also provide more detailed information.
• Upgradeability. Are the system’s components (i.e., lasers, detectors, filters and software) upgradeable? Tool providers offer many entry-point options for their customers, but research is a dynamic process, so you want to make sure your system can be altered or customized at any time to meet your lab’s needs.
• Throughput and time to results. Instrument and reagent manufacturers are continually improving the time to results. Throughput on different flow systems is tied to the sample vessel (i.e., tubes, bottles or microplates). The instrument’s automation options, and their effect on throughput and time to results, can also influence the selection of a system.
• Lab footprint. Standard benchtop instruments continue to be the standard in most labs and core facilities. However, an increasing number of portable systems (which perform a range of applications) have been introduced by various tool providers (e.g., MilliporeSigma, BD Biosciences, Beckman Coulter, Cell Signaling Technology and Miltenyi Biotec, Thermo Fisher Scientific).
• Software. Tool providers typically bundle software and training tutorials with their instruments. There are a variety of vendors that provide more detailed analysis software (e.g., FLOWJO) A number of kits are available for data analysis that researchers can access online.
• Technical support. When selecting an instrument, the initial decision making can be overwhelming. But make sure to ask your tool providers what sort of support is available after you have purchased their instrument or reagents.
• Price. Consistently, price is the foremost factor influencing purchasing considerations. Pricing for flow systems can span a wide range depending on the number of features and components selected for the system. Researchers should consider the total cost per experimental run, and the frequency with which cell separation and characterization experiments will be performed. This expense of owning your own system, along with the frequency of experimental runs, should be compared to the cost to have samples analyzed in a core facility.

Summary

Despite its advanced age, flow cytometry and cell sorting technology continues to evolve with the development of improvements at all levels—not only in the specific components of the instrumentation but also in reagents (i.e., dyes, assay kits and antibodies) and software used for protocol development and data. Instruments such as the CytoFLEX from Beckman Coulter are providing a smaller lab footprint and portability options for researchers without compromising the performance and features typically found in larger instruments. Methodologies such as RNA fluorescent in situ hybridization (FISH) are being integrated into flow analysis with reagents like ebiosciences’ (now part of Thermo Fisher Scientific) PrimeFlow™ RNA assay. Flow cytometry is being coupled with mass spectrometry—mass cytometry or CyTOF which is run on the Fluidigm’s Helios system.

Together, these evolving pieces are enabling researchers and clinicians to further expand their understanding of the biology, characteristics and regulation of cells at the single-cell level (DNA, RNA and proteins) as well as within populations. What was once a core lab purchase has quickly become something affordable to individual labs; it can even function in the most minimal of field settings.

Thus, with plenty of functional and mechanical overlap between the many offerings, the decision of which platform to buy largely reflects what you as an individual researcher or as a community need to accomplish today and hope to attempt tomorrow. The opportunities just keep proliferating.

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