Flow Cytometry Buying Guide
Buying a new flow cytometer is a huge investment, and with the extensive variety and diversity of flow cytometers on the market, it is often difficult to ensure you purchase the instrument that best fits your needs.
This guide highlights key considerations for purchasing new instrumentation in order to help you make the right decision when choosing new flow cytometry equipment for your lab.
Flow cytometry is a powerful technique that simultaneously measures and analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. Flow cytometry is widely used for analyzing expression of cell surface and intracellular molecules, characterizing and defining different cell types in heterogeneous cell populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence (Figure 2).
Over the past 30 years, flow cytometry has become an indispensible tool for many scientific researchers and clinicians. The evolving technology ensures its application in a number of fields, including cell biology, drug discovery, cancer research, neuroscience, stem cell research, pathology, immunology, hematology, plant biology, food science and marine biology.
FLOW CYTOMETRY TECHNOLOGY AND GENERAL CONSIDERATIONS
A flow cytometer is made up of three main systems (Figure 2). The fluidics system transports particles in a sample stream to the interrogation point and takes away the waste (Figure 1). The optics system consists of lasers to illuminate the particles in the sample stream and optical filters to direct the resulting light signals to the appropriate detectors. The electronics system converts the light signals from the detectors into electronic signals that can be processed by the computer. For some instruments equipped with a sorting feature, the electronics system is also capable of initiating sorting decisions to charge and deflect particles.The Fluidics System
The fluidics system within the flow cytometer transports particles in a fluid stream to the laser beam for interrogation (Figure 1). When a sample in solution is injected into a flow cytometer, the particles are randomly distributed in a three-dimensional space. For accurate data collection, it is important that the particles or cells are passed through the laser beam one at a time. Most flow cytometers accomplish this by injecting the sample stream into a stream of faster flowing sheath fluid within the flow chamber. The flow of the sheath fluid accelerates the particles and restricts them to the center of the sample core, so that a single stream of particles is created. This process is known as hydrodynamic focusing (Figure 1).
Figure 1. Hydrodynamic focusing within the fluidics system of the flow cytometer
The Optics System
After the process of hydrodynamic focusing, each particle passes through one or more beams of light (Figure 2). Light scattering, through a forward scatter channel (FSC) or fluorescence emission (if the particle is labeled with a fluorochrome), provides information about the particle’s properties. Cell granularity or internal structural complexity is provided via a side scatter channel (SSC). The laser and the arc lamp are the most popular light sources in modern flow cytometry.
Fluorescent measurements taken at different wavelengths can provide quantitative and qualitative data about fluorochrome-labeled cell surface receptors or intracellular molecules, such as DNA and cytokines (Figure 2). The argon ion laser is most commonly used in flow cytometry. This is because the 488 nm light that it emits excites more than one fluorochrome.The Electronics System
Once a cell or particle passes through the laser light, emitted side scatter and fluorescence signals are diverted to the photomultiplier tubes (PMTs) and a silicon photodiode collects the forward scatter signals (Figure 2). The number of detectors will vary according to the instrument and its manufacturer. All of the signals are routed to their photodetectors via a system of mirrors and optical filters. Optical filters control the specificity of detection by blocking certain wavelengths, and transmitting others.
The photodetectors convert the light signals into electronic signals, which are converted into a voltage pulse for plotting on a graphical scale. Log amplification is normally used for fluorescence studies and linear scaling is preferable where there is not such a broad range of signals, e.g. in DNA analysis.
The measurement from each detector is referred to as a ‘parameter’, e.g. forward scatter, side scatter or fluorescence. The data acquired in each parameter are known as the ‘events’ and refer to the number of cells displaying the physical feature or marker of interest.
Figure 2. Schematic overview of a typical flow cytometer setup
Modern flow cytometers can analyze several thousand particles or cells every second. When you purchase a flow cytometer for quick data acquisition times, it is important to ensure that the resolution and data quality are not compromised by these rapid sampling rates. Pre-enrichment of target cell populations can aid in acquiring higher-quality data.
Flow cytometric data is stored according to a standard format, the flow cytometry standard (FCS) format, which was developed by the Society for Analytical Cytology.
The data generated by flow cytometers can be plotted in a single dimension to produce a histogram, in two-dimensional dot plots, or even three-dimensional plots. An important principle of flow cytometry data analysis is to selectively visualize the cells of interest while eliminating results from unwanted particles e.g. dead cells and debris. This procedure is called gating. A gate is a numerical or graphical boundary that can be used to define the characteristics of particles to include for further analysis.
Manual gating is often considered to be a time consuming and highly subjective process. Recent progress on automated population identification using computational methods has offered an alternative to traditional gating strategies. Specific gating protocols exist for different applications, thus the choice of software is an important consideration in flow cytometry data analysis.
Today, most instruments use software for instrument control, data acquisition, analysis and display. Many instruments provide software control for many or all instrument hardware functions, including fluidics, lasers, electronics, and sorting. Further, operations such as start-up and shutdown sequences, cleaning cycles, calibration cycles and self-test functions can be monitored and regulated by software, so that instrument operation can be simplified and optimized for the user.
When purchasing a flow cytometer, it is imperative to consider the ease-of-use of the system. This is, in part, determined by the system’s software. You should 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 you can perform more complex analyses in the future.
It is important to investigate how regularly the analysis software is updated, and whether you will be able to receive automatic upgrades. You should consider whether the software will integrate with hardware you already have in the laboratory. In addition, you should determine how the software will present the data to you and establish whether this meets your needs.
It is important that the damaging external effects of light exposure, temperature variation and mechanical agitation are minimized to the sample under investigation. This will ensure that sample and, therefore, data integrity is maintained. Consideration should also be given to the suitable selection of sheath fluid so that cells can be analyzed and/or sorted with minimum disturbance.
Most flow cytometers can utilize sample volumes ranging from several microliters to hundreds of microliters. If you have a small volume of precious cells, you may wish to consider using a microfluidics-based flow cytometry chip. The development of microfluidic, lab-on-a-chip (LOC) technologies is one of the most innovative and cost-effective approaches toward the advancement of cytometry.
Some flow cytometers are now fitted with a magnetic pre-enrichment column, which is particularly useful when studying rare cells. The Attune® Acoustic Focusing Cytometer, from Applied Biosystems, has been used to improve rare-event detection in human circulating endothelial cells.
Automated introduction of samples to the flow cytometer allows for walk-away operation to be performed on large numbers of samples, using a variety of formats, from single test tubes to multi-well plates. Single tubes are often contained within rectangular grid arrangements or carousel formats to match laboratory equipment used for sample preparation. The Vi-CELL® XR Cell Viability Analyzer by Beckman Coulter has been proven as an automatic and cost-effective means to perform the trypan blue dye (see section on fluorochromes) exclusion method for counting dendritic cells.
The need for high throughput of varying samples is dependent on the number of lasers, and affected by the number of ways you want to sort your cells, for example, into two, four or six sorted populations. The more sorted groups you have, the more you need to think about where they will be placed in your sample vessels. Typically, flow cytometers sort cells into sample tubes or microplates of up to 384 wells. While it is possible to run single samples on most flow cytometers, the ability to use microplates makes higher throughput possible.
CLINICAL APPLICATIONS OF FLOW CYTOMETRY & KEY CONSIDERATIONS
Flow cytometry has become an important component in the diagnosis and monitoring of patients with a diverse array of diseases.
Immunophenotyping is used to study protein expression in cells and for disease diagnosis. The technique is used primarily in the diagnosis of leukemia. Clinical laboratories use immunophenotyping panels to label white blood cells with a specific range of antibody markers directed against surface proteins associated with specific cancer cells. By doing this, clinicians can determine which malignant cells are present in a sample and give an accurate patient diagnosis. Immunophenotyping can be carried out on blood, spinal fluid and bone marrow samples.
Flow cytometry is important for the detection of minimal residual disease (MRD) in bone marrow, a leukemia relapse marker, which is beyond the limit of morphological detection using conventional microscopy. Multiparameter flow cytometry methodology using the Beckman Coulter FC500 Series Flow Cytometer has been shown to detect low levels of the leukemia-associated phenotype in acute myeloid leukemia patients.
Figure 3:Multiparameter flow cytometry using the FC 500 Series Flow Cytometer from Beckman Coulter.
In a clinical laboratory, flow cytometry often plays an import role in solid organ transplant procedures. For example, flow cytometry can be used to crossmatch a recipient's serum with donor lymphocytes to detect antibodies that could interfere with engraftment. After the organ transplant, analysis of the peripheral blood lymphocytes may help to indicate early rejection and bone marrow toxicity during immunosuppressive therapies, and to help in the differentiation of infections from transplant rejection.
Other current clinical applications of flow cytometry include detection of autoantibodies, diagnosis of cancer and immunodeficiency disorders, monitoring of HIV infection, measuring the efficacy of cancer chemotherapy, quantifying fetal maternal hemorrhage, stem cell and reticulocyte enumeration, platelet counting and quality control.
RESEARCH APPLICATIONS OF FLOW CYTOMETRY & KEY CONSIDERATIONS
The ability to simultaneously measure multiple parameters on a cell by cell basis is probably the most powerful aspect of analytical flow cytometry. This allows research scientists in all industries to use flow cytometry for a wide range of applications, with many institutes and companies having a dedicated flow cytometry core. Research applications of flow cytometry include:
Cell Cycle Analysis
One of the most common applications of flow cytometry is measurement of the DNA content of cells. In this way, different treatments can be assessed for their effect on cell cycle distribution; for example, it is used in drug screening to discover compounds that affect the proliferation and growth of cells. The technique can easily be applied to both mammalian and yeast cells, although due to their much smaller size and DNA content, yeasts show less defined phases of the cell cycle. There are many different fluorochromes that can be used to determine DNA content, each having differing excitation and emission wavelengths.The CyFlow® Cube 8 from Partec offers analysis of thousands of cells per second, with DNA staining reagents for ploidy analysis, genome sizing, cell viability and cell cycle measurements.
Protein Expression Analysis
Flow cytometry can accurately quantitate reporter gene expression (for example, Green Fluorescent Protein, GFP) in each cell in a population being transfected. Co-transfection of a reporter plasmid and a reference plasmid can be very variable in normal human cells, making interpretation difficult in reporter gene assays. However, using flow cytometry, reporter and reference plasmid expression can be quantitated at the single-cell level, even in cases of low transfection efficiency. In addition, heterogeneity in reporter expression and transient effects in gene expression can be examined. An example of this can be seen in this study on plant cell ploidy using the compact, personal BD Accuri C6 Flow Cytometer from BD Biosciences.
Figure 4: BD Accuri C6 Flow Cytometer
Apoptosis and necrosis can be distinguished by flow cytometry on the basis of differences in morphological, biochemical and molecular changes occurring in the dying cells. Apoptosis occurs via a complex signaling cascade that is tightly regulated at multiple points, providing many opportunities to evaluate the activity of the proteins involved. The initiator and effector caspases are particularly good targets for detecting apoptosis in cells and their detection by flow cytometry has become widespread. Flow cytometry is a popular technique for studying Natural killer (NK) cells, a subset of lymphocytes involved in innate immunity, in vitro and in vivo. In this study, a novel flow cytometry kit is used to characterize and detect NK cell-mediated killing of human K562 myeloma cells in human peripheral blood using the EMD Millipore Guava easyCyte™ Flow Cytometer.
Figure 5: Guava easyCyte™ Flow Cytometer
Cell Proliferation Assays
The flow cytometer can measure cell proliferation by labeling resting cells with a cell membrane fluorescent dye, carboxyfluorescein succinimidyl ester (CFSE). When the cells are activated, they begin to proliferate and undergo mitosis. As the cells divide, half of the original dye is passed on to each daughter cell. By measuring the reduction of the fluorescence signal, researchers can calculate cellular activation and proliferation. Successful cell proliferation analysis by dye dilution requires sensitive instrumentation and an extremely bright dye to accurately distinguish fluorescently labeled cells from autofluorescence after several cell divisions.
Intracellular Calcium Flux Cells interact with each other and their environment through signal transduction pathways. When these pathways are activated, membrane-bound calcium ion channels pump calcium into the cell and rapidly increase the intracellular calcium concentration. The cytometer can monitor the flux of calcium into the cell and measure the extent to which cells respond to the stimuli. The most common method is to use the UV-excited dye Indo-1, which fluoresces at a different wavelength when it is bound to calcium than when it is not.
A cell sorting cytometer interrogates and characterizes each cell as it passes through the laser. If a cell or particle can be specifically identified by its physical or chemical characteristics, it can be separated using a cell sorter. The sorter uses sophisticated electronics and fluidics to identify and separate the cells of interest from the fluidic stream into a collection vessel.
Many flow cytometers can now be upgraded to perform this function known as fluorescence-activated cell sorting (FACS™), or electrostatic cell sorting. The light scattering and fluorescent characteristics of each cell are compared to criteria programmed into the chosen instrument. If there is a match, the fluid stream is charged as it exits the nozzle of the fluidics system. When the droplets pass through an electrostatic field, they are deflected in different directions based on their charge. The speed of flow sorting depends on several factors, including particle size and the rate of droplet formation. A nozzle producing 30,000-100,000 droplets per second is ideal for accurate sorting.
Imaging Flow Cytometry Imaging 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.
Imaging flow cytometry combines the statistical power and fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy. The Cellometer from Nexcellom is a great example of effectively combining microscopic imaging and cytometry methods with comparable detection sensitivity.
Imaging flow cytometry can be used to study cell function morphology, internalization, cell signaling, co-localization, cell death, stem cell differentiation and cell-cell interactions, to name but a few. The technique is also a good fit for clinical applications by providing a convenient means for imaging and analyzing cells directly in bodily fluids, such as blood and urine.
You should consider the extent of the technical support and training that is available from the manufacturer at the point on purchase. It is also recommended that you look into the company’s policy on after sales service. While everything is operating well, this may be the last thing on your mind, but in the event that after sales support is required, it is important to know how will this be achieved.
You will need to carefully evaluate how much of your laboratory space can be dedicated to your new instrument. The good news is that flow cytometer manufacturers appreciate that laboratory space is extremely valuable and consequently are trending towards smaller, more compact systems, (see Figure 4). Benchtop-friendly systems that do not compromise on function are becoming more readily available. Personal flow cytometers, which are commonplace on the market today, tend to have an extremely compact footprint and are often portable for field work and for the diagnosis and monitoring of diseases such as HIV/AIDS, leukemia, tuberculosis and malaria.
The issue of biosafety is attracting increasing attention in the field of flow cytometry. Risks are similar to those faced in other biological laboratories dealing with hazardous solvents, infectious agents and blood products. The particular challenges with flow cytometry are the high pressures applied to samples and the aerosols that can be associated with sorting. A solution to this problem is provided in this study using the Coriolis®μ air sampler system from Bertin Technologies.
In order for a system to grow with your changing requirements, you will need to investigate the potential for upgradability. For example, you may wish to increase the number of lasers and detectors to enable multiple labeling and facilitate more targeted population studies. Will you be able to expand your instrument, or perhaps trade it for a more flexible model?
Another extremely important factor that you will need to consider when buying a flow cytometer is its compatibility. In order to reliably assess the suitability of your potential new flow cytometer, you may wish to investigate its compatibility with automated or robotic lab systems, with LIMS or other data storage systems, as well as with reagents and consumables.
As highlighted throughout this guide, the most powerful aspect of flow cytometry is the fast and simultaneous multiparametric analysis of proprieties of single cells. Sophisticated flow cytometers, capable of analyzing signals of up to 18 fluorochromes at the same time, are available today. It is becoming routine to do five- to seven-color experiments without any major difficulties. Advancements in flow cytometer instrumentation have spurred the development of new fluorochromes, also known as fluorophores, and antibody conjugates that take advantage of these capabilities.
The difficulty in choosing the best suited fluorochromes for any given application means we often stick with fluorochrome combinations that have been historically used in our labs, rather than the ones that are best suited for the given application. While panel design can be a complex process, speaking with other scientists and manufacturers should enable you to better optimize the results of your flow cytometry experiments.
Tandem dyes are being used more frequently in flow cytometry. They consist of two conjugated dyes that are covalently linked and in close proximity (30-50 nm). When one of the fluorochromes is excited, the energy is transferred to the nearby fluorochrome, which in turn produces fluorescence emission. The process is called FRET (fluorescence resonance energy transfer). It is a clever way to achieve higher Stokes Shifts and, therefore, increase the number of colors that can be analyzed from a single laser wavelength. Tandem dyes should be used with caution however; see Table 1 below.
There are a few basic rules that you should consider when choosing a fluorochrome or a combination of fluorochromes. Table 1 below summarizes what to consider.
Type and number of lasers
Affects whether the optical system can excite a given fluorochrome and detect a combination of fluorochromes
Optical system design
Affects dye detection efficiency
Instrument settings, e.g. PMT voltage
Affects dye detection efficiency
Optical filters choice
Affects brightness and spill over background
Choose a color in each excitation range before considering a second color for the same laser
Affects the compensation between different colors
Choose the brightest fluorochromes that can be used on your instrument
Choose the brightest fluorochrome for the least well expressed protein/antigen and the dimmest fluorochrome for the most well expressed protein/antigen
Choose fluorochromes with emissions having the least spectral overlap. Use an online spectra viewer
Every fluorochrome has a wide fluorescence emission spectrum that extends beyond the narrow window of light allowed by an optical filter for a specific fluorochrome
Use tandem dyes with caution
Tandem dyes are prone to uncoupling and photobleaching. Use a single-stained control for any tandem dye in order to verify that the tandem has not uncoupled
Table 1: Considerations for purchasing flow cytometer fluorochromes
THE FUTURE OF FLOW CYTOMETRY
The demands on clinical diagnostics, clinical research, phenotypic research and drug development have driven flow cytometry technology since its first introduction in the 1960s. However, the major breakthroughs in the past few years are: ‘load and go’ technology – the ability to load a machine and walk away, and portability or compactness of instruments.
Although not yet authorized for use in the clinical setting, the AQUIOS CL Clinical Cytometer from Beckman Coulter is the first fully automated, authentic ‘load and go’ cytometry system. If cleared for in vitro use by the United States Food and Drug Administration, the cytometer will provide efficient testing for routine applications such as immunophenotyping.
Recently a new platform that couples flow cytometry with mass spectrometry has been developed. This technology, known as mass cytometry, offers a new approach in which antibodies are tagged with isotopically pure rare earth elements, rather than fluorophores, allowing simultaneous measurement of greater than 40 parameters, while circumventing the issue of spectral overlap. Mass cytometry is very much in its infancy, but has the potential to become an important tool for drug discovery and clinical applications in the future.
One major limitation in conventional flow cytometry is that hydrodynamic focusing is normally required to align single cells in a laminar flow, which prevents this technology from being used for quantitative in vivo detection. However, the nascent field of in vivo flow cytometry (IVFC) has begun to yield results in applications such as monitoring circulating cancer cells (CTCs). IVFC has the capability to measure the dynamics of fluorescently labeled CTCs continuously and non-invasively. Studies to date have shown that IVFC has higher specificity and sensitivity than conventional flow cytometry methods. IVFC have been used to investigate several cancers including leukemia, prostate cancer, breast cancer, hepatocellular carcinoma and melanoma, among others. In the future, IVFC may be an important tool for elucidating mechanisms that drive metastasis and may be used to monitor the efficacy of cancer therapy. With advances in the technique, IVFC could potentially be used clinically in both cancer diagnosis and treatment.
The high speed quantitative high content analysis of cells also makes flow cytometry an attractive technology for drug discovery applications and it is currently used at many stages of this process. Recently it is also finding a niche in drug screening laboratories, sharing bench space with other high throughput technologies. Flow cytometry is becoming an ideal tool, particularly in an environment where primary cell-based assays are increasingly being deployed to monitor drug responses.
The potential of flow cytometry as a drug screening platform was first realized upon the introduction of plate-based sampling on flow cytometers. High throughput flow cytometry is still at ‘the lower end’ of the capacity scales in a typical drug screening laboratory, where one to two million compounds can be profiled in just a few weeks, but further improvements are currently in progress, such as 1536-well sampling.
Flow cytometry is increasingly recognized as an invaluable technology in biomarker research. Owing to its multiparametric nature, it can provide highly detailed information on any single cell in a heterogeneous population. Its versatility means it can be conducted in both the preclinical and clinical setting, generating biomarker data that can drive decisions pertaining to dose selection in clinical trials, to treatment options for cancer sufferers and even suitability of patients to receive transplants. In a recent study, published by the FDA, biomarker research has been combined with flow cytometry methods for analyzing clinical trial outcomes.
The versatility of flow cytometry means that it remains at the forefront of clinical and all research-based applications for the future.
Whether you are a first time buyer of flow cytometry equipment, or an experienced user, there are a number of factors to consider if you are choosing a new platform. It is also essential that you thoroughly examine your current and your future application needs. For tips to get the best out of your cytometer, read the SelectScience article Flow Cytometry: Five Technical Tips by Camilo Moncada, PhD, Director of Quality Control at Rockland Immunochemicals.
Visit the SelectScience product directory for an overview of the latest flow cytometry technology from leading manufacturers and read user reviews. Keep up-to-date with the latest cytometry methods by visiting the SelectScience application note and video libraries.
Drug Discovery & Development Editor