2012 CYTO

All Tutorial sessions will be offered on Saturday, June 23, 2012 at the Congress Center Leipzig. A separate fee is required to attend these sessions. The list of planned sessions is provided below.

Multi-parametric single cell assays are a key strength of flow cytometry as a research methodology. Tremendous power to interrogate and analyse complex cell populations has been derived through the development and application of polychromatic ("many colour") flow cytometry. While polychromatic flow has grown increasingly more accessible and is no longer limited to a handful of labs its power still brings complexity and challenge.

This tutorial is designed to provide a brief overview of the key components of polychromatic flow cytometry. Particular attention will be paid to the advantages (and pitfalls) of performing polychromatic assays on the new class of instruments that are equipped with large numbers of lasers (e.g. 5-10 lasers). The value of these "polylaser" instruments in other applications,including the multiplexing of fluorescent proteins, will also be discussed.

Topics to be covered include instruments (lasers, detectors, filters, setup and monitoring), reagents (fluorochromes and conjugates), controls, and panel design. After attending this session participants should gain (i) a basic understanding of the principles of polychromatic and polylaser flow, (ii) an appreciation of the issues they need to consider and address in order to be successful at polychromatic flow, (iii) an appreciation of the power of polylaser instruments and (iv) some pointers of where to go for help when they run into problems.

This tutorial will review the current state of the art in the identification and isolation of hematopoietic progenitor cells, epithelail stem cells, multipotent mesenchymal stem cells, adipose stem cells, endothelial precursors and pericytic stem cells. We will address several questions such as: What markers have been used to detect stem cells in normal human bone marrow, epithelial and adipose tissues? Do markers define populations with biologically unique properties? Must a self-replicating cell be rare or multipotent to be considered a stem cell? What is the difference between a stem cell and a progenitor cell?

Continuing advancements in fluorescence labeling, microscope sensitivity and environmental conditions under the microscope are enabling live cell microscopy to be standard practice in biological research. Experimental results now reveal a new dimension of cellular dynamics at the tissue level and molecular dynamics at the cellular level. The tutorial will provide practical strategies for confocal and multi-photon microscope imaging of living cells, tissues and whole organisms. Methodology will be stressed using numerous research examples. Basic topics include: minimizing light exposure, choosing the objective lens, maintaining sterility, autofocusing, incubator microscopes. More advanced techniques include fluorescence recovery after photobleaching and super-resolution optical microscopy.

Attendees of the course will understand how to prepare live cell samples for fluorescence microscopy, how to operate the microscope for obtaining high quality images while minimizing damage to the sample, and become familiar with more advanced and emerging techniques for molecular analysis in cells.
Although this tutorial covers the basics, attendees are also recommended to take the pre-congress introductory course on Image and Flow Cytometry.

Functional super-resolution (fSR) microscopy is based on the automated toponome imaging system (TIS). fSR-TIS
provides insight into the myriad of different cellular functionalities by direct imaging of large subcellular protein
networks in morphologically intact cells and tissues, referred to as the toponome. By cyclical fluorescence imaging of at
least 100 molecular cell components, fSR-TIS overcomes the spectral limitations of fluorescence microscopy, which is
the essential condition for the detection of protein network structures in situ/in vivo. The resulting data sets precisely
discriminate between cell types, subcellular structures, cell states and diseases (fSR). With up to 16 bits per protein, the
power of combinatorial molecular discrimination (PCMD) is at least 2100 per subcellular data point. It provides the
dimensionality necessary to uncover thousands of distinct protein clusters including their subcellular hierarchies
controlling protein network topology and function in the one cell or tissue section. The technology enables the
researcher to directly detect so called lead proteins controlling the precise assembly of proteins in cell membranes in
situ/vivo, having paved the way for the detection of rearrangement rather than regulation of proteins as an essential
mechanism controllung cell function in health and disease. fRS-TIS has layed the ground for next–generation
biomarkers, disease-specific drug-targets, and the systematic mapping of the human toponome on a proteome-wide
scale (ISAC best paper award 2008).

High throughput screening is now a real opportunity with flow cytometry. It is now possible to collect thousands of samples per day in a highly ordered way. However, designing and preparing these assays does require some careful attention to details and automated prep becomes an important component. One advantage is that you only sample very small volumes (like 1 uL) instead of 50 to 250 uL. This means instead of needing far more cells to start, you can actually get away with fewer than you might think. This tutorial will discuss all aspects of HT flow cytometry and it will also discuss high content flow cytometry where you might have a very large number of samples as well as a large number of parameters.

After participating in this tutorial, the student will be more aware of the advantages and possible disadvantages of having to work in an automated, or semi-automated régime. They will have a good knowledge of how to approach the preparation, and the analysis of data. They will also learn some analytical tools that have been designed particularly for high throughput and high content analysis.

This tutorial will introduce basic level students to the fundamental concepts of measuring Signal Transduction Pathways using intracellular analysis of phospho-epitopes in conjunction with analysis of cell surface (CD) epitopes. Methodological concepts will be stressed, including cell preparation, antibody validation and data analysis. For the intermediate/advanced student, the tutorial will cover methods useful in monitoring signal transduction pathways in peripheral blood and bone marrowl samples, focusing on leukemias, including the concept of “real-time” monitoring responses to molecular targeted therapies.

At the end of this tutorial, the student will understand critical technical details that impact the flow cytometric measurement of signal transduction pathways in conjunction with cell surface epitopes needed to identify different cell populations. Specific technical areas will be addressed, including the impact of different fixation and permeabilization techniques on cytoplasmic and cell surface markers. We will discuss the application of signaling cytometry in the analysis of complex cell populations, including human bone marrow.

Detailed syllabus/agenda:
The still poorly explored world of microbial functioning is about to be uncovered by a combined appliance of older and new technologies. Especially bacteria are still in the dark both in the view of their phylogenetic affiliation as well as their metabolic capabilities and functions. Both individuality and diversity lead to the complex and concerted actions of microbial consortia. The tutorial gives an overview of the current state of the art within the field. It indicates practice handling of microbes from the very beginning (meaning sampling, and detachment and fixation procedures) and goes on to discuss pitfalls and profit when analyzing cells without any further treatment. There will be a wide discussion on how to treat vital and fixed cells by applying structural and functional related fluorescent probes und goes on to cell sorting applications which open the door to the “omics” technologies. Flow cytometry will be understood as the mediator technology to obtain a deeper insight into the heterogeneity of populations and functioning of microbial communities.

Experience level of students:
The students should have a basic experience in flow cytometry. They should know the principles of cytometric measurement, staining and different applications in cell and microbial biology. A basic knowledge in biology and/or microbiology sciences would also be useful. Welcome are also scientists who want to combine microbial single cell techniques with Omics-technologies.

Cell sorting on jet-in-air sorters is an essential technique for isolation of cellular subpopulations, but it poses inherent risks of aerosol exposure. Thus, performing an accurate risk assessment of the hazards associated with sorting in a specific laboratory is an important aspect of sort laboratory management and safety assurance. With the increased variability in samples subjected to sorting and in instrument configurations and attachment options creating a Standard Operating Procedure (SOP) for sorting of unfixed cells that contains the proper safety elements can be difficult. This tutorial will provide guidance in this process.

Following the steps outlined and using the resources provided, laboratory directors and managers should be able to develop a SOP that includes safeguards to be applied during sorting that are appropriate for the work conducted and the specific sort system set-up.

This tutorial will cover introductory image and data analysis for cytometry and high-content screening, including image preprocessing, segmentation of objects in images, and capturing per-cell measurements, as well as the ensuing data analysis for both simple and complex phenotypes, using gating or training of classifiers using human guidance, respectively, and the interpretation of results in an screening setting. The tutorial will include examples using CellProfiler and CellProfiler Analyst.

After this tutorial, participants should have a grasp of the basics of image and data analysis for high-content screening, with sufficient background to begin to use CellProfiler or other tools for image and data analysis.

Flow cytometric methods to assess cell growth and death will be reviewed with an emphasis on how to select the appropriate assay, the technical details necessary for assay success, and combining multiple assays for multiparametric analysis of both cell growth and death. The tutorial will take a very practical approach, and actual cytometry data will be analyzed as part of the program. Related topics including cell tracking, detection of early DNA damage and autophagy will also be covered.

After completing the Tutorial, the student should be able to:

(1) Have a working knowledge of flow cytometric assays for proliferation, apoptosis and autophagy.

(2) Be able to select an appropriate proliferation or apoptosis assay for their experimental system,
procure the necessary reagents and carry out the assay.

(3) Combine proliferation and apoptosis assays with each other and with other cytometric assays, including

immunolabeling.

Cell sorting is often one of the fundamental steps in the purification of specific cells from mixed parent populations. This technology has found widespread application in the biological sciences in areas as diverse as plant biology, marine sciences, immunology research and neuroscience. With this broad range of applications, and users with varied scientific backgrounds are seeking greater understanding of the technology and how it is, or may be, applied.
This tutorial aims to cover the principles of electrostatic cell sorting followed by an update on recent publication utilizing cell sorting. Participants with little knowledge of sorting should hopefully learn something about the technology, while more experienced practitioners should receive a basics refresher with exposure to recent work in the field.

Tutorial Session XIII

Detection of Circulating Tumor Cells: Challenges and Perspectives

Klaus Pantel, Department of Tumor Biology, University Medical Center Hamburg, Hamburg, Germany

Solid tumours derived from epithelial tissues (carcinomas) are responsible for 90% of all new cancers in Europe and the main four tumour entities are breast, prostate, lung and colon cancer. Present tumour staging is mainly based on local tumour extension, metastatic lymph node involvement and evidence of overt distant metastasis obtained by imaging technologies. However, these staging procedures are not sensitive enough to detect early tumour cell dissemination as a key event in tumour progression. Moreover, the detection of CTC in the peripheral blood of cancer patients holds great promise as companion diagnostics in clinical trials testing novel drugs (“companion diagnostics”). This course will provide an overview and critical discussion of sensitive assays that allow the specific detection of single tumour cells or small amounts of cell-free tumor DNA in the peripheral blood of cancer patients. These methods allow the detection and characterization of early metastatic spread and will provide unique insights into the biology of metastatic progression of human tumours including the effects of therapeutic interventions. This course will focus on the detection and further characterization of individual CTC and circulating cell-free DNA as “real-time liquid biopsy” helping to identify therapeutic targets and potential resistance mechanisms to therapy, which might contribute to the development of improved individualized targeted treatment of cancer patients.

This tutorial is aimed at newer or prospective Core Facility managers and staff. We will cover the factors that need to be considered when setting up a new facility and expanding a current one. This includes financial and budgetary considerations, practical operational issues, marketing and growing the core, how to judge success and future planning. Attendees should leave with an appreciation of the most important factors and a checklist of the practicalities of running a core.

This tutorial will present an overview of imaging cytometry modalities by comparing solid state (slide based) and flow cytometry instrumentation. Emphasis will be on the use of cultured cells and tissue samples that were labeled using immunohistochemistry and/or immunofluorescent methods. Participants will become knowledgeable of the critical steps involved in sample preparation, reagent selection, event segmentation and data analysis.

SCIENTIFIC TUTORIALS

About ISAC