SATURDAY, 18 MAY, 2013
12:30 – 14:00 Tutorial Session 1
Apoptosis, Autophagy and DNA Damage
Bill Telford, National Cancer Institute, Bethesda, MD, USA
In the first part of the Tutorial (Telford), flow cytometric methods to assess apoptosis will be thoroughly reviewed. Students will learn how to select the appropriate assay, the technical details necessary for assay success, and combining multiple assays for multiparametric analysis of cell. The tutorial will take a very practical approach, and actual cytometry data will be analyzed as part of the program. Autophagy, a phenomenon both related to and distinct from cell death will also be covered.
In the second part of the Tutorial (Darzynkiewicz), detection of DNA damage by flow and image cytometry will be covered, both as a component of apoptosis and as a means of analyzing the effects of pharmacological, toxicological and environmental insults to live cells. Detection of both DNA damage itself and of phosphoproteins critical for DNA organization and structure that serve as markers for DNA damage will be particularly emphasized.
After completing the Tutorial, the student should be able to:
(1) Have a working knowledge of flow cytometric assays for apoptosis, autophagy and DNA damage.
(2) Be able to select an appropriate apoptosis and DNA damage assay for their experimental system, procure the necessary reagents and carry out the assay.
(3) Combine multiple apoptosis assays to develop powerful systems for studying the complex signaling and progression of apoptosis.
Tutorial Session 2
Cytometer Performance Characterization and Standardization
Robert Hoffman, Consultant, Livermore, CA, USA
Cytometers make optical measurements of particles. Using flow cytometers as the primary example, this tutorial will provide an understanding of the measurement process and how to critically and objectively characterize the instrument’s ability to make fluorescence and light scattering measurements. The use of standard instrument performance criteria and testing materials can allow minimal performance requirements to be established for an assay and assure that results on different instruments in different laboratories get comparable results when running the same sample. Authoritative standards (traceable to a standards setting organization) are generally not available for cytometry. Students will learn about the use and limitations of commercially available standard materials and some options for laboratory prepared standards.
Tutorial Session 3
Approaches in Image-Based High Content Screening
Susanne Heynen-Genel, Sanford Burnham Medical Research Institute, La Jolla, CA, USA
Jeffrey Price, Vala Sciences Inc. and Sanford Burnham Medical Research Institute, La Jolla, CA, USA
This tutorial will cover current approaches in image-based high content screening and provide examples. After attending this tutorial, the participant should have a good understanding of the currently available image-based screening methods and their advantages and limitations.
Tutorial Session 4
Biosafety: Risk Assessment and SOP Development
Kevin Holmes, National Institutes of Health, Bethesda, MD, USA
This tutorial will provide the attendee with an overview of biosafety principles, as they apply to flow cytometry with emphasis on cell sorting. The process of risk assessment for determination of biosafety containment levels and the development of a Standard Operating Procedure (SOP) for flow cytometers will be discussed.
Tutorial Session 5
High Throughput and High Content Screening Flow Cytometry
J. Paul Robinson, Purdue University, West Lafayette, IN, USA
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 may actually be able to run far more samples than you might think. We will discuss several different implementations of HT flow cytometry and the types of assays that are usable in this format. This tutorial will also discuss aspects very high content flow cytometry where you might have a very large number of samples as well as a large number of parameters such as might be found in CyTOF data where parameters may exceed 20-30 per assay.
After participating in this tutorial, the participant 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.
14:15 – 15:45 Tutorial Session 6
Proliferation Tutorial: Cell Cycle Progression and Division - Unraveled by Flow Cytometry
Paul Wallace, Roswell Park Cancer Institute, Buffalo, NY, USA
This tutorial will consider flow cytometric techniques for monitoring different aspects of cell proliferation. After a brief review of cell cycle biology, we will discuss:
1) Principles, methods and analysis strategies for measuring cell cycle progression using DNA binding dyes, thymidine analogues, proliferation-related antigens and phase specific expression vectors;
2) Principles, methods and analysis strategies for monitoring extent of cell division using proliferation tracking dyes (CFSE, PKH26 and newer analogs of each).
Examples of applications using the different methods will be used to compare and contrast the information that is obtained using different probes and techniques. We will end with an opportunity for participants to discuss their real world problems and invite you to send your challenging cases to one of the instructors for discussion during the tutorial.
After participating in this tutorial attendees should have a clear understanding of 1) events that must occur in order for a cell to enter the cell cycle and to divide; 2) the difference between cell cycle progression and cell division as measures of proliferation; and 3) advantages and limitations of specific flow cytometric methods commonly used to used to quantify cell cycle progression and/or extent of cell division.
Tutorial Session 7
Cell Sorting: Fundamentals, Applications and Troubleshooting
Geoff Osborne, University of Queensland, Queensland, Australia
This tutorial will provide information on the fundamentals of electrostatic cell sorting and an overview of the application of cell sorting in practical situations. Attendees will discuss some of the areas where things can and do go wrong, and what steps can be taken to improve sorting experiments.
Tutorial Session 8
Image Quantification and Analysis using CellProfiler
David Logan, Broad Institute, Cambridge, MA, USA
Image-based experiments using cultured cells have proven to be a powerful means of generating information-rich data for biological applications. This tutorial will instruct biologists in the concepts and application of CellProfiler, an open-source, freely-downloadable software package designed for large-scale, automated phenotypic image analysis. We will work through hands-on examples, including construction of an analysis pipeline, optimization of module settings, automatic cellular feature detection and measurement, and exporting results. We will also briefly discuss the basic principles of supervised machine learning in order to score phenotypes where phenotypic differences between samples are subtle and/or complex. The tutorial participant should gain a working knowledge of CellProfiler and how to process and analyze their high- or low-throughput, high-content experiment.
Tutorial Session 9
Analysis of Receptor Dynamics Using Flow Cytometry
Alexandre Chigaev, University of New Mexico, Albuquerque, NM, USA
Yang Wu, University of New Mexico, Albuquerque, NM, USA
This tutorial will cover two subjects:
Part I Real-time flow cytometry of integrin signaling: lessons learned
Have you ever dreamed of performing signaling experiments on live cells, in real-time, at natural receptor abundance, and without long-term pre-staining of cells using fluorescent dyes? We have been doing these for more than a decade. A unique property of integrin molecules is the ability to rapidly change ligand binding affinity, to “stand-up” on the cell surface, and to respond to the ligation of the binding pocket through the changes of the molecular conformation. These changes generate a plethora of molecular conformations, which, at the cellular level, are translated directly into different modes of cell-adhesive behavior. G-protein coupled receptors, tyrosine kinase receptors, and other signaling pathways (including nitric oxide/cAMP pathway) rapidly regulate integrin conformation, and modulate cell adhesion and mobilization, by triggering so-called “inside-out” signaling pathway. In the current presentation we focus on the basic methods and novel unpublished results including FRET-based measurement of molecular extension of LFA-1 integrin, as well as rapid de-activation of VLA-4 integrin through previously undescribed signaling pathways. By the end of this section participants will not only learn about real-time flow cytometry, experimental design, and interpretation of the data, they will also understand the relationship between integrin molecular conformation and immune cell adhesive behavior (rolling, firm adhesion, or cell detachment). We will also discuss practical questions related to probe design, and commercial sources of existing probes.
Part II High Throughput Flow Cytometry in Drug Discovery
Now you’ve learned all the basics about monitoring surface protein signaling in real time, it’s time to move on to the hot field of drug discovery. In the second half of the tutorial, we will introduce you to a newly developed approach that is not only suitable for the real-time analysis of receptor trafficking, but also compatible with high-throughput flow cytometry (HTFC). The later application demonstrates that in addition to measuring individual samples from test tubes, flow cytometry has the capability to collect data from 40 samples per minute, and thus contribute to the early stage of drug discovery. In the sample approach, a newly available reporter protein (FAP) tag was fused with target protein, and ligand induced receptor trafficking was measured by flow cytometry in real-time. We will share with you some basics of HTFC and the FAP reporter system, experimental design of HTFC compatible assays, associated data analysis, and work flow. Videos taken in our high-throughput flow cytometry center at the University of New Mexico will also be available to demonstrate the automated processes of the sample plate preparation and screening. By the end of the second half of this tutorial, we hope you will get some idea about the role of high-throughput screening in the field of drug discovery, gain some experience on setting up assays suitable for high-throughput flow cytometry, be familiar with a new type of biosensor, and bring home with some thoughts and ideas about how FAP and HTFC can do for your research.
Tutorial Session 10
Evaluation and Purchase of an Analytical Flow Cytometer: Some of the Numerous Factors to Consider
Robert Zucker, U.S. Environmental Protection Agency, Research Triangle Park, NC, USA
Nancy Fisher, Univeristy of North Carolina, Chapel Hill, NC, USA
1) When purchasing a flow cytometer, the decision of which brand, model, specifications, and accessories may be challenging. The decisions should initially be guided by the specific applications intended for the instrument. However, many other factors need to be considered, which include hardware, software, quality assurance, support, service, and price and recommendations from colleagues. These issues are discussed to help guide the purchasing process. 2) Student should obtain information that will be used to evaluate different factors that are neccesary to evaluate in purchacing a new flow cytometer.
16:00 – 17:30 Tutorial Session 11
Seeing a More Colorful World: A Guide to Polychromatic Flow Cytometry
Pratip Chattopadhyay, National Institutes of Health, Bethesda, MD, USA
This tutorial will cover the latest tips and tricks for successful polychromatic flow
cytometry experiments. Participants will leave the session with a practical,
working knowledge of how to develop or optimize the technology for their
Tutorial Session 12
Analysis and Sorting of Rare Cell Populations
Phil McCoy, National Institutes of Health, Bethesda, MD, USA
This tutorial will address how to find the proverbial 'needle in the haystack' when needing to identify or sort rare populations of cells. In the contemporary flow cytometry laboratory, there is often the need to accurate identify rare cells, such as circulating endothelial cell, endothelial progenitor cells, tumor cells, or immune subpopulations such as plasmacytoid or monocytoid dendritic cells.The student will gain an appreciation of obstacles in the accurate identification of rare cells and of strategies to overcome these obstacles and to assure better experimental data.
Tutorial Session 13
Quantitative FRET Microscopy
Gyorgy Vereb, University of Debrecen, Debrecen, Hungary
The use of fluorescence (Förster) resonance energy transfer (FRET) for assessing molecular interactions in cellular systems is exponentially expanding. Several methods, mostly for microscopy, have been proposed, but most of them that made it to broad use owed to their simplicity suffer from being only qualitative or even from being prone to misinterpretation of results. The educational outcome of the tutorial is to provide the audience with stable foundations for applying a simple, yet qualitative FRET procedure that can be performed in any commonly available laser scanning fluorescence microscope. Addition outcomes include skills in interpretation of FRET data, and a broader knowledge on the pros and cons of various FRET methods that allow an educated choice of the approach most appropriate for the biological question.
Tutorial Session 14
Growing a Cytometry Facility: Adding Value with Hardware and Education
Derek Davies, London Research Institute, Cancer Research UK, London, United Kingdom
Alfonso Blanco, Conway Institute of Biomolecular and Biomedical Research, Univeristy College Dublin, Dubline, Republic of Ireland
Core facilities are now common in all work settings. Flow cytometry is a well-established technique but the core faces particular challenges in the face of expanding technology. In particular, cores need to bring added value to their users and institutional setting. But how can core facility staff keep up with the latest developments, how can they receive appropriate continuing education and how can this be passed on to users of a facility? We will discuss evaluation of technology and strategies for importing this into a core and also how education on site, at relevant meetings and by remote learning can benefit the facility. At the end of the tutorial the delegate will be aware of the approaches that can be taken to bring added value to the core, its staff and its users.
Tutorial Session 15
Cell Organizer: Building Models of Cell Structure from Microscope Images and Using them for High-Content Screening and Cell Simulations
Gregory Johnson, Carnegie Mellon University, Pittsburgh, PA, USA
Devin Sullivan, Carnegie Mellon University, Pittsburgh, PA, USA
CellOrganizer is an open source software system that can learn models of the size, shape and spatial distribution of cellular components directly from images. These models are generative, which means that they can be used to synthesize new images of cells that are statistically similar to the ones they were trained on. Such images are useful for testing image analysis algorithms, and can be used as the basis for spatially-realistic cell simulations using systems such as Virtual Cell and MCell. Perhaps most importantly, CellOrganizer models represent a transportable means of representing the results of High Content Screening (HCS) assays that is not dependent on a specific instrument, assay or cell type. This tutorial will focus on how to use CellOrganizer and how to interface it with other software.
The tutorial will begin with a brief overview of the conditional structure of the models within CellOrganizer and the system organization. The first part of the tutorial will focus on training generative models. Students are strongly encouraged (but not required) to bring a laptop. Attendees are also encouraged to bring a fluorescent cellular image dataset of their own to use for building a model, but datasets will be available at the tutorial for attendees who do not have one. Ideally, images should be two or three dimensional single cell images (i.e., already segmented) with different fluorescence channels for a fluorescently labeled target protein (ideally a protein showing a punctate or vescular pattern), a cell membrane or cytosolic-labeled marker, and a DNA marker (but these are not strict requirements). The second part will focus on synthesizing cell images from the models and importing the images or model parameters into other software systems. The last part will focus on adding new capabilities to the open source system, such as modules for building new types of components.
Students should leave this session with mastery of the principles behind building probabilistic models from images and practical experience with training and using them with CellOrganizer. They will be able to use them to compare results from different HCS assays using the generative model parameters, and import synthetic images into cell simulation systems.