November 15, 2009 (Vol. 29, No. 20)

GEN recently spoke to several scientists with expertise in the flow cytometry arena about emerging trends and additional potential applications for this versatile research tool. Clare Rogers, application scientist at Accuri Cytometers; Robert Balderas, vp, biological sciences at BD Biosciences; T. Vincent Shankey, Ph.D., principal staff advanced research scientist, advanced technology/cellular analysis business group at Beckman Coulter; Fred Koller, Ph.D., president and CEO of Cyntellect; and Jason Whalley, flow cytometry manager at Millipore share their knowledge and predictions for the future in this flow-cytometry roundtable.

GEN What have been a few of the major advances in flow cytometry for life science research over the past five to ten years?

Clare Rogers The increased availability of reagents, along with advances in flow cytometer design, has improved access to the technology for the average life science researcher. The choices in kits, buffers, antibody-fluorochrome conjugates, and protocols make designing and implementing multicolor assays easier than ever.

The common use and proliferation of digital instruments has greatly improved efficiency. Instruments that use high-resolution, digital-signal processing and have expanded dynamic range (six decades of fluorescence as opposed to the standard four) allow researchers to separate data acquisition from analysis by obviating the need for setting voltages. This advance allows novice flow cytometrists to be able to collect high-quality data with minimal direction or supervision.

Lastly, the proliferation of multiplex bead-based assays has allowed flow cytometers to replace time-consuming, commonplace assays such as ELISAs, and opened the door for flow cytometry to be used much more widely in the fields of gene and protein microarray analysis.

Robert Balderas Over the past decade, flow cytometry has made cell sorting and analysis easier and more accessible to life science researchers working across a wide range of advanced applications—allowing them to get more data from every experiment via a variety of color choices. All the while, this platform has grown significantly in terms of its capabilities to address a breadth of applications. As new extracellular and intracellular proteins are identified and their respective antibodies conjugated for flow cytometry, the utility of flow cytometry will only increase. 

Today’s flow cytometry instruments incorporate a wide array of technologies—including advanced fluidics, lasers, and optical detection systems—and workflow improvements that make these instruments powerful tools for cellular analysis that are approachable to a new generation of researchers and clinicians. 

T. Vincent Shankey, Ph.D. Current flow-cytometry instrumentation allows simultaneous measurements of up to 17 independent color parameters. This increase is the result of new low molecular weight and tandem dyes, and nanocrystals that have been developed in concert with small, relatively inexpensive solid-state lasers with emission lines from far red to the UV.

GFP-linked gene reporter systems, developed by Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien, have allowed cell biologists to track movements of proteins in living cells, and to identify and isolate cells expressing one or multiple gene products.

Digitization of signals from photomultiplier detectors allows acquisition of multiple types of signals (integral, peak, log, and linear) from one channel and application of accurate compensation after signal acquisition.

Software advances facilitate faster and more comprehensive analysis of complex, multiparameter experiments and compensation during data analysis, and enable modeling of complex cell populations based on qualitative or quantitative expression of multiple markers.

Flow-cytometry instrumentation has been developed that provides acquisition of fluorescence signals from individual flowing cells in conjunction with individual cell images. This allows localization of targets inside individual cells, enumeration of individual spots (e.g., fluorescence in situ hybridization probes), and identification of different types of cells based on individual cell morphology.

MHC tetramers allow detailed analysis and understanding of the immune system’s response to well-defined antigens. This is highly useful in understanding the response to viral and other pathogens, and in the development of vaccines to these pathogens.

Fred Koller Flow cytometry is based on fluidics systems and, therefore, cells must be in fluid suspension to be analyzed on the instrument. The user must remove the cells from the plate or flask, typically by enzymatic disruption or scraping, and resuspend the cells prior to analysis. For this reason, flow cytometry systems have historically analyzed one sample at a time, making the speed of the systems too slow for primary drug screening projects. Recent advancements in cytometry now allow the user to analyze the cells right where they are grown—in situ, in plates, and flasks—by label-free (brightfield) assays or by fluorescence assays, at high speeds.

Jason Whalley Even though flow cytometry has been available for many years, use within research labs has not reached its full potential. The invention of microcapillary flow technology has launched a new generation of powerful, benchtop systems. The technology eliminates the need for complicated fluidics and sheath fluid, and allows for the development of instruments that fit onto the lab bench. These new instruments, combined with intuitive, application-specific software and validated, ready-to-use reagent kits, have brought instruments to the lab bench with similar performance as those in the core lab.

GEN For which new or emerging biotech applications might flow cytometry be appropriate?

Clare Rogers Flow cytometry should emerge as an instrument of choice to study human disease and health states by measuring cell populations based on surface marker expression, and by utilizing emerging flow techniques to measure gene and protein expression, molecular-level changes in phosphorylation and methylation states, and to screen for rare cell types. Flow cytometry is gaining wider application in microbiology and environmental sciences, as well.

For these applications to be realized fully, scientists need a small, low-cost, flexible, high-performance instrument and the ability to use automation to process many samples quickly.

Robert Balderas The development and characterization of new antibody specificities and conjugates for flow cytometry are helping to advance scientists’ understanding of cell biology, as well as enabling significant progress in stem cell and cell therapy research. Flow cytometry, with its multicolor, multivariate platform capability, offers a research tool that can isolate cells of interest from the millions of other cells present. This capability may advance discoveries and perhaps one day lead to exciting therapeutic breakthroughs, such as cell therapy.

In recent years, an explosion in our knowledge of stem cells and cell therapy is expected to take us to new heights of personalized medicine and targeted efficacy.  Along with these breakthroughs, new quality and safety systems will be needed to ensure patient safety and reduce risk.  As research moves toward clinical trials and ultimately new cell therapies, researchers will look to companies with experience in delivering quality GMP products and services to provide them with the tools and systems to transform disease treatment and management.

T. Vincent Shankey, Ph.D. Flow cytometry is an important methodology in cellular analysis, and the range of tests in which it is used will continue to grow. Significant and representative application include the characterization and identification of normal and cancer stem cells, monitoring signal transduction pathways (Phos-flow or signaling cytometry), and high-complexity/multiplexed bead arrays for simultaneous quantification of hundreds of different analytes.

Fred Koller Flow cytometry is a well-established method for cellular analysis. This technology has been widely implemented in clinical labs for analysis of blood cells (nonadherent cell types) as well as in cellular research laboratories for adherent and nonadherent cells. It is now being applied to biological assay development, providing new cell-based assays for detecting any toxic effects of compounds under investigation, and validating hits from compound screens in drug discovery.

Jason Whalley Two of the most exciting emerging applications are cell signaling and stem cells. These two areas can greatly benefit from the analysis of markers or targets at the single-cell level instead of analysis of mixed populations. New cell-signaling reagent kits simultaneously detect key protein-activation states and cross talk using directly conjugated, phospho-specific antibodies; while new stem cell reagent kits provide rapid, sensitive assessments of embryonic and neural stem cell phenotypes at various stages of differentiation.” 

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