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Dec 1, 2013 (Vol. 33, No. 21)

Spectral Flow Cytometry Makes Debut

  • “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

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    Scientists at the BIO5 Institute simultaneously sorted green fluorescent protein (GFP)-positive and GFP-negative nuclei and used microarrays to determine which transcripts were most abundant in the GFP-positive nuclei. They were able to determine the cell-type-specific expression patterns of 12 genes that were selectively expressed in the phloem of their plant model.

    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

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    To relieve cell-sorting processing bottlenecks, laboratories might consider more affordable, easy-to-use cell-sorting units. Bio-Rad asserts that its S3 flow cytometer is designed to make cell sorting more accessible.

    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.

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