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Feature Articles : Dec 1, 2013 (Vol. 33, No. 21)

Spectral Flow Cytometry Makes Debut

  • Kathy Liszewski

Although flow cytometry was invented more than 45 years ago, it continues to grow and expand into new areas that marry it with other modern cutting-edge technologies.

Harnessing the power of the full spectrum, coupling it with mass spectrometry, and utilizing it for expression profiling are a few of the new and intriguing applications heralding the next generation of flow cytometry.

Recent advances in optics and detectors are allowing a new technology, spectral flow cytometry, to make full spectral measurements on the sub-millisecond scale. John Nolan, Ph.D., principal investigator at La Jolla Bioengineering Institute, describes his approach: “There has been a longstanding interest in measuring the complete emission spectra from individual cells. We’ve developed the instrumentation to do this in a robust and routine way.”

Dr. Nolan says the new technology differs in several important aspects from conventional flow cytometry. “Instead of utilizing photomultiplier tubes, dichroic mirrors, and band-pass filters to measure cell-derived fluorescence at specific wavelengths, spectral flow cytometry uses prisms or gratings to disperse light over a detector array for high-speed, wavelength-resolved detection. Spectral unmixing, a data-analysis approach that estimates the amount of each fluorescent probe in the mixture spectrum, replaces compensation, which is required in conventional flow cytometry to account for fluorescence spillover between channels.”

Spectral Flow

Although autofluorescence can be a significant problem in typical flow cytometry, spectral flow easily resolves the issue. “With spectral flow, autofluorescence is just treated as another ‘color’ and can be resolved from other colors using spectral unmixing, effectively reducing a major source of background,” notes Dr. Nolan.

Dr. Nolan and colleagues demonstrated proof of principle by using calibrated beads stained with six different quantum dots to demonstrate the analytical performance of the instrument. To evaluate performance in a typical immunophenotyping application, they analyzed peripheral blood mononuclear cells stained with canonical surface markers used to discriminate lymphocyte subsets.

“Spectral flow was clearly able to resolve CD14+ monocytes, CD3+ T cells, and CD4+ and CD8+ subsets,” says Dr. Nolan. “This shows that spectral flow cytometry has a dynamic range suitable for such conventional applications.”

Dr. Nolan and colleagues, aware that the emergence of spectral flow cytometry opens the doors to using many different types of fluorescent and other optical probes, are tackling other challenges for this evolving technology. “At present, commercial options are designed for the conventional flow cytometry paradigm of one color per detector,” remarks Dr. Nolan. “Within 5 to 10 years, we will likely see development of new probes, improved instrumentation, new analytical software, and new applications that take advantage of the spectral flow cytometry approach.”

Mass Cytometry

A new technology adapts the analytical capabilities of atomic mass spectrometry to address the challenges of multiparametric flow cytometry. Advancing this technology is CyTOF®, a mass cytometry system developed and produced by DVS Sciences. Emulating flow cytometry, this technology involves tagging antibodies with isotopically pure rare-earth metals rather than fluorophores.

After incubation with a panel of metal-conjugated antibodies, hundreds of cells per second are passed through an argon plasma that atomizes and ionizes the metal tags that are subsequently analyzed by a time-of-flight mass spectrometer. In a typical cell analysis experiment, the data derived in less than four minutes of raw collection provides analysis of more than 100,000 cells.

“The CyTOF system has the capability to measure more than 100 different parameters with negligible signal overlap,” notes Scott Tanner, Ph.D., CTO and co-founder. “Currently, we offer 34 distinct metal tags and continue to develop more,” adds Nicole Ellis-Ovadia, head of marketing. Researchers can label their own antibodies or choose from a catalog of over 200 pre-labeled antibodies or application-specific panel kits.

With the huge amount of data generated by mass cytometry, one key challenge is high-dimensional data analysis. According to Dr. Tanner, “Mass cytometry requires novel multivariate analytical solutions. While our data files (standard .fcs format) are compatible with all flow analytical platforms, our preferred solution is DVS Cytobank, a new platform that provides conventional flow data workflow as well as validated and emerging unsupervised protocols designed for high dimensional data.”

Applications for mass cytometry continue to grow and include basic research on cell genesis and functionality, drug discovery (including screening samples with limited availability), and translational applications such as in the clinical diagnostics arena. Dr. Tanner says, “All of these areas share a huge demand for high dimensional, single-cell analysis and will also benefit from application-specific hardware, reagents, and software solutions.”

Single-Cell Transcriptomics

In the last five years, highly multiplexed, single-cell gene expression studies have revealed a great degree of heterogeneity in cell populations thought to be relatively uniform. However, little work has been done in immunological systems, where heterogeneity is known and expected, according to Mario Roederer, Ph.D., a senior investigator at the NIH. Dr. Roederer’s team performed a series of studies to define the gene signatures and identify the coordinate expression patterns of multiple genes in unique cell subsets.

“Our goal is to marry the power of protein expression analysis via flow cytometry with gene expression profiling of single cells to interrogate peripheral blood mononuclear cells,” says Dr. Roederer. “Combining these technologies also provides information as to the post-transcriptional regulation in these cells, something that neither technology alone can do.”

Dr. Roederer’s team utilizes a multicolor, fluorescence-activated, cell-sorting system along with Fluidigm’s BioMark™ system to dissect single-cell gene expression. The latter is performed on 96 samples simultaneously and can measure 96 or more genes in each sample. “Although the technology has been used in several disparate biological settings, methodological details for optimal and quantitative application were lacking,” comments Dr. Roederer.

“We devised methods to qualify primers for efficient quantitative cDNA amplification as well as to account for the wide variation in heterogeneous mRNA species in immunological cells. We showed that primers do not compete in highly multiplexed amplifications. We also determined that the limit of detection is a single mRNA transcript.”

The team examined the activation of CD4+ T cells at the single-cell level and identified subsets in the population. They showed that co-expression of certain gene combinations such as CXCR5/CCL5 and DPP4/TlA1 were actually rare events rather than common occurrences as previously thought when bulk mRNA was investigated.

Dr. Roederer says that the technology could be applied to vaccine studies: “We can utilize this approach to look at gene families and examine how the functional state differs in order to assess if vaccines induce a protective response or not. There is a wealth of information at the multiple-cell level (pooled arrays) as well as at the single-cell level.”

Sorting Nuclei

Separating, sorting, and profiling different cell populations from complex multicellular eukaryotic organs can be challenging. David W. Galbraith, Ph.D., who has a research program housed within the BIO5 Institute at the University of Arizona, notes that, “Prior to gene expression measurements, it is critical to separate out different cell types. The challenge has been not only to reliably purify those cells of interest from tissue, but also, at the same time, minimize disruption of normal cellular function during isolation.”

Enter fluorescence-activating sorting of nuclei. Dr. Galbraith and his team adopted a different approach: “Instead of focusing on the cell, we concentrated on the transcriptional center of the cell, the nucleus. We initially explored this strategy using the plant model, Arabidopsis. We produced transgenic plants expressing a histone2A-green fluorescent protein (GFP) fusion that selectively labeled the nucleus. Following gentle homogenization of the plant tissues, we employed flow cytometry and fluorescence-activated sorting to purify the nuclei from different cell types.”

The team simultaneously sorted GFP-positive and GFP-negative nuclei and used microarrays to determine which transcripts were most abundant in the GFP-positive nuclei. “We were able to determine the cell-type specific expression patterns of 12 genes that were selectively expressed in the phloem, the vascular tissue of plants,” says Dr. Galbraith. “Further, we were able to show that profiling mRNA within the nucleus accurately represents mRNAs in the cytoplasm.”

According to Dr. Galbraith, the team is now extending this paradigm to mammalian cells and tissues. By using a promoter-specific regulatory sequence and this particular marker targeted to the nucleus, the team is able to extend their analysis to many different types of cells.

In addition, investigators can make use of the many different colored fluorescent proteins that are becoming available. “For example, a mouse transgenic for combinations of fluorescent proteins targeted to different brain cells would lead to a more sophisticated and comprehensive understanding of microanatomy, physiology, and regulation of cell-type specific gene expression in that organ,” remarks Dr. Gailbraith. “Furthermore, there are also many ways to translate this technology to the study of human disease.”

Robust and Economical Cell Sorting

Cell sorting is one of the fastest and most accurate methods for isolating individual cells or a population of cells from a complex mixture from both eukaryotic and prokaryotic cells. Cell-sorting instruments typically sort cells expressing fluorescent proteins or labeled with specific markers.

“Cell sorting can also be used to isolate single cells for subsequent single-cell qPCR or digital PCR studies,” says Melissa Ma, global product manager for the cell biology business unit at Bio-Rad Laboratories.

Cell-sorting systems are usually located in core facilities at academic, government, and industrial research institutions not only because of their high cost ($250,000 to $1 million), but also their complexity, which requires the need for a dedicated operator. As more and more researchers are utilizing the power of cell sorting, core labs can sometimes experience a bottleneck, with investigators waiting for their turns at sorting. Because of these issues, companies are seeking to develop more affordable and easy-to-use units. An example of this emerging trend is Bio-Rad’s S3™ cell-sorting system.

“We developed a more economical, highly automated, and easy-to-use cell sorter to address the needs of the customer,” notes Ma. “The S3 allows researchers to sort cells at high speeds while maintaining sensitivity and purity. Because it is easy and straightforward to use, core facilities can teach end users how to sort their own samples in a minimal amount of time. This not only alleviates backlogs, it maximizes core use and enhances efficiency. At the same time, individual labs are able to afford and run the units themselves, with no dedicated personnel required.”

Isolation of cells from complex mixtures sometimes requires two or more fluorescent labels, necessitating that cell sorters be equipped with more than one laser. “The S3 includes one or two lasers (a single 488 nm laser, or 488 and 561 nm lasers) enabling up to four-color detection along with forward- and side-scatter detectors,” remarks Ma. “Another important feature is automation. The S3 can be automatically set up and calibrated in less than 30 minutes with minimal user intervention.”

Affordable and user-friendly systems will likely make cell-sorting instrumentation more accessible to a variety of new labs and core facilities that could not previously afford or support a cell sorter.