Meeting Report: The Leading Edge in Cytometry

Bad Nauheim was in early autumn splendor as cytometry leaders, researchers, and vendors met to conduct and participate in the Leading Edge in Cytometry Meeting at the Max Planck Institute for Heart and Lung Research (MPI-HLR) in September 2016.
The meeting covered less familiar technology, such as the DEPArray™, Mass Cytometry and the “unknown” e.g. the single cell printer; and included miscellaneous lectures that covered uncommon applications.
From the interaction and discussions that took place at the meeting it became evident that there was a keen interest in promoting an efficient transfer of knowledge between cytometry professionals and researchers, and also between companies and everybody else.

 Introduction, nanoparticles and the complexity of immunological memory

The meeting was opened by Prof. Stefan Offermanns (MPI-HLR) who extended a warm welcome to all the participants. While highlighting the broad use of cytometry at the MPI he put particular emphasis on the purpose of the meeting as a platform for facilitating a comprehensive exchange of information.

The first speaker was Angelina Georgieva (MPI-HLR) who introduced us to skeletal muscle stem cells. These stem cells reside as satellite cells on myofibers and express numerous well-defined markers. Angelina presented her research on the self-renewing properties that enable muscle repair. Working with both wild type and reporter mice she clarified her sample preparation protocol for cell sorting. She highlighted the need for sample pre-enrichment, a strategy to cope with auto-fluorescence, and presented the outcome of subsequent downstream assays such as western blot, qPCR and ChIP-seq.
Nanoparticles are of particular interest as carriers for drug delivery in therapeutic procedures.

Against this background Volker Mailaender (MPI-Polymer Research. Mainz) demonstrated that labeling with iron-containing particles provides the possibility to track cell fate in vivo by using noninvasive magnetic resonance imaging (MRI). Using flow cytometry he emphasized the impact of different types of nanoparticles on functionality and differentiation in stem cells: showing discrepancies between CD marker expression and differentiation marker genes at the RNA level depending on the type of nanoparticles used.

The keynote speaker was Hyun-Dong Chang. At present he is president of the DGfZ; and also the scientific head of the flow cytometry core facility of the DRFZ in Berlin. Hyun-Dong’s presentation focused on the analysis of immunological memory by flow cytometry. Highlighting the complexity of immunological memory he expanded on a study comparing memory Tcells from human bone marrow and peripheral blood, revealing that memory T cells in the bone marrow rest in terms of proliferation,
transcription, and migration despite expressing CD69, which is usually indicative of early activation.

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DEPArray, zebrafish and Single Cell Sorting for Next Generation Sequencing

The first of the technology sessions began with the DEPArray™ (Menarini Silicon Biosystems). Applications specialist Hichem Gallala gave an overview of the technology. Cell sorting is based on fluorescence imaging and capturing target cells within microfluidic cages by di-electrophoretic forces. Limitations on this instrument are mainly confined to the number of cells that can be loaded onto the system.

The use of the DEPArray™ as an efficient scientific tool to analyze and isolate circulating tumour cells (CTCs) was validated by Rui Neves who is a member of the CANCER-ID team at University Hospital Dusseldorf. CTCs are clinically significant and can act as biomarkers for monitoring systemic cancer. He explained that molecular characterization of CTCs could be a relevant guide to individualized cancer therapies. However, CTCs are found at very low frequencies and as such enrichment procedures and cell sorting technology are crucial to downstream assays needed for molecular characterization. Because of the heterogeneous nature of CTCs it is essential to characterize single CTCs. He described a workflow for obtaining pure CTCs using CellSearch® and subsequent single cell capture by the DEPArray™.

The zebrafish system is used to gain insight into organogenesis and hence also used for the study of pancreas development. Carol Yang (MPI-HLR) introduced her research into the role of autonomic innervation of the endocrine pancreas during early zebrafish development. She presented the protocols required for the analysis and isolation of pancreatic islet cells for subsequent transcriptome profiling. Carol also demonstrated the use of index cell sorting for enhancing the isolation of intact pancreatic islets, and is planning to use the DEPArray™ in the near future to improve the quality of islet cell isolation for single cell transcriptome profiling.

The final speaker of the day was Wieland Keilholz (BD) who presented innovative solutions for single cell transcriptomics and NGS library preparations. He demonstrated various options in terms of reagents and instruments – e.g. the FACS Melody cell sorter for single cell isolation and the Precise multiplexed assays for single cell RNA sequencing.

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Mass Cytometry, principles and application

The morning was dedicated to mass cytometry; a cutting-edge domain known and widely discussed in the cytometry field, but at the same time more enigmatic to those of us who have yet to benefit from the experience of working with this technology. In order to provide a comprehensive overview of mass cytometry the meeting was organized to include (1) an overview of the technology, (2) a research focus with data acquisition, and (3) data analysis strategies.

The technology and underlying principles were introduced by Andrius Serva, an applications specialist at Fluidigm. Apart from giving a detailed description of the technology he imparted background information on the use of metal tagged probes, panel design and bar coding.
From here Andreas Gruetzkau – group leader of the Immune Monitoring Group at the DRFZ (Berlin) – took the lead. Within the context of his research on single cell profiling in chronic-inflammatory rheumatic diseases he revealed the intricacies of multi-parameter panel design and workflows for mass cytometry, including algorithms for data analysis and visualization. Andreas presented a comparison of phenotyping by mass cytometry versus traditional flow cytometry – he clarified the output data from the instruments and disclosed the similarity of the results. He underlined the importance of high-parameter cytometric profiling for new biomarker discovery in order to obtain a better classification and understanding of inflammatory diseases.

Advancing onwards Antonio Cosma – who leads the FlowCyTech core facility at the CEA, France – picked up the challenge of engaging us in the complexities of analyzing high-throughput multi-dimensional data. Antonio emphasized the need for robust computational tools to aid analysis. He demonstrated that combining tools – such as viSNE and SPADE – enables the visualization of data in multiple ways,permitting a more accurate and critical analysis of data sets. With ease he guided us step-by-step through SPADE analysis extracting and organizing the meaningful content of combinatorial exploding data. He clarified how this algorithm clusters cells into populations and then projects them into a tree exposing interconnected clusters of subpopulations, thereby revealing underlying hierarchies and cellular heterogeneity.

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Single Cell Printing and Single Cell Analysis (and education in between)

Continuous education in cytometry is seriously challenged by rapidly developing technology and new applications. Elmar Endl (University Hospital Bonn) tackled this subject with the skill of an experienced core facility manager; reminding us that we develop our training and teaching skills without any formal instruction. Elmar candidly spoke about his own experiences in the field and emphasized the need for maintaining communication and interaction in the cytometry community. He introduced us to cytometry.de – a platform where members are given many options for communicating, learning, writing and sharing documents.

The G-protein mediated signaling pathway was introduced by Denise Tischner (MPI-HLR) as she presented her work on the role of GPCR expression in neuro-inflammation. Flow cytometry and cell sorting were used to identify sub-populations of immune T cells and endothelial cells during naive and inflammatory conditions. Expanding on the analyses of specific GPCR expression she highlighted the use of the Fluidigm C1 system that enables the capture of single cells, the analyses of 192 genes/cell by RT- PCR and the identification of specific subpopulations which are characterized by a specific GPCR expression pattern.

A novel way of cell sorting was demonstrated by Andre Gross (Cytena, Freiburg) who introduced the Single Cell Printer. This instrument combines a piezoelectric drop formation with an imaging system for the precise isolation of single viable cells. Using various cell types and results from collaborators he showed pure single cells could be cloned and that viability and function were intact after sorting. There were some pertinent questions of a 3D printing possibility with cells – this was not ruled out.

The final talks were dedicated to large particle analysis. George Dubelaar (Cytobuoy b.v.) introduced the CytoSense which covers a particle size range up to 1.5 mm in diameter. Apart from the usual fluorescence features this instrument contains an imaging system. It also acquires multiple data points per particle thereby also quantifying the number and distribution of smaller fluorescent entities within a particle. These features are critical for the classification of phyto-plankton species and the analysis of filamentous algae.

Daniela Ehgartner (Vienna University of Technology) expanded on the above topic by presenting her studies on the viability and morphology of filamentous fungi. She used the CytoSense for viability staining and the quantification of metabolically active spores. The flow cytometry data combined with clear imaging details of spore germination and hyphae formation not only proved the CytoSense to be ideal for the understanding of fungi development processes but also, for any kind of large particle analyses.

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Summary

The meeting was organized with the goal of promoting and enhancing professional competence in the present-day cytometry research environment. This was easily achieved by the speakers who -while showing their dedication to their own cytometry related field – effortlessly provided a platform for the exchange of information generating strong interest and deliberation. A wealth of information was gained opening up different aspects of cytometry and approaches across disciplines. Personal contacts for networking were encouraged and accepted for future support, feedback and resources.

Ann Atzberger

Flow Cytometry Service manager Max-Planck-Institute for Heart and Lung Research and Experimental Medicine · Flow Cytometry