Flow Cytometry
Collapse Integrating Flow Cytometry and Single-Cell Transcriptomics

Mario Roederer, Ph.D. will be presenting the webinar Integrating Flow Cytometry and Single-Cell Transcriptomics. Mario serves as Senior Investigator on the ImmunoTechnology Section, Chief on the Flow Cytometry Core, and Chief on the Nonhuman Primate Immunogenicity Core. The immune system is comprised of incredibly diverse sets of cells, each programmed to carry out overlapping sets of effector functions. Quantifying any one function provides an incomplete view of the immune response, as information about what other responses are generated is absent. Quantifying multiple responses is far superior, but when carried out on a bulk level, loses information about cellular heterogeneity, gene programs, and a myriad of interactions that may occur at the single-cell level. Since individual cells are the atomic unit of immune function, the maximum information content is achievable only by measuring these functions independently and simultaneously on a cell-by-cell basis. For this reason, flow cytometryis a powerful technology to assess immune function in settings like vaccination and pathogenesis. Nonetheless, current flow cytometrytechnology is limited to measuring the expression of ~16 proteins per cell. To extend the multiplexing of gene expression measurements, we now combine single cell sorting (based on cell surface phenotype) with highly-multiplexed qPCR for 96 or more genes. We choose to quantify lymphocyte-centric genes, including those encoding transcription factors, signaling molecules, effector;molecules, and regulatory molecules. On a single-cell basis, we can correlate protein expression with gene expression. Discordant results for the same gene reveal post-transcriptional regulatory mechanisms. We have identified gene signatures associated with vaccine-elicited T cells as well as with productively SIV-infected cellsin vivo. This technology gives us an unprecedented view into the complexity and range of immunological functions expressed by vaccine or virus-specific immune cells. Using this approach, we can search for correlates of clinical outcome based on either: quantitative gene expression; and/or cell subset representation, enumerated by groups of cells sharing gene expression profiles. These analysesgive us new insights into functional immune states in pathogenesis, treatment, and vaccination.

Formats Available: Streaming
Original Seminar Date: February 19, 2014
On-Demand Release Date: Available Now

Approved Credit:
  • ISAC: 1 hour ICCE
  • ASCP: 1 hour CMLE

  • Topics & Pricing InformationTopics & Pricing Information Integrating Flow Cytometry and Single-Cell Transcriptomics
    Collapse Flow Cytometry Biosafety Course

    Authors: Kevin Holmes, Steve Perfetto, Hank Pletcher, Ingrid Schmid

    The Flow Cytometry Biosafety course will provide a summary of biosafety principles as they apply to flow cytometry and cell sorting and provide an overview of applicable standards and risk assessment. The topics covered will be:

    • Biosafety Principles and Procedures
    • Understanding Risk Assessment
    • Instrument-specific Standard Operating Procedures (SOPs)
    • Biosafety in Cell Sorting
    • Containment Testing

    At the completion of this course, students will have a clearer understanding of the principles and practices of biosafety as it pertains to flow cytometry, in particular cell sorting. Additionally, you will have a list of resources to aid in risk assessment and the development of Standard Operating Procedures in your own lab. 

    To view the course, Click "Register Now."

    If you are not logged in to the CYTO U website, you will be directed to a new page asking for your login information.  
     
    For current ISAC members, your user name and password to CYTO U are the same user name and password you have for the members-only section of the ISAC Web site. If you cannot remember your password, you can re-set it on the ISAC Web site by clicking on the "Forgot Password?" link on the log-in page.
    Formats Available: Streaming

    Approved Credit:
  • ISAC: 1.50 hours ICCE
  • ASCP: 1.50 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information Flow Cytometry Biosafety Course
    Collapse Designing, Planning, and Performing Polychromatic Flow Cytometry Experiments (2014 Scientific Tutorial)
    Polychromatic flow cytometry provides the power to analyze, in great depth, the complex relationships between cellular subsets and function.  We will discuss the latest hardware, reagent, and data analysis considerations for performing successful polychromatic experiments, and provide a practical roadmap for developing and improving this technology.  This year, there will be a new focus on executing large studies successfully, including unique aspects of planning and data analysis.
    Formats Available: Streaming

    Approved Credit:
  • ISAC: 1.50 hours ICCE
  • ASCP: 1.50 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information Designing, Planning, and Performing Polychromatic Flow Cytometry Experiments (2014 Scientific Tutorial)
    Collapse Algorithmic Analysis of Flow Cytometry Data (Part 1) (2014 Scientific Tutorial)
    This two-session tutorial will demonstrate the automated analysis of flow cytometry data using five different approaches.  Traditional manual gating analysis suffers from poor reproducibility and objectivity, and is very difficult for high-dimensional data.  Several algorithms have now been developed, using different approaches that may be more suited to different datasets and biological questions.  Five approaches will be highlighted in this tutorial, covering a range of approaches from just identification of targeted or unknown populations, to a full pipeline of data analysis.  The tutorials will consist of brief demonstrations of data analysis by each of the approaches.  After the tutorials, the students should have an appreciation of the power of automated analysis, the requirements for entering their data into an automated analysis system, and how to choose the most appropriate analysis method for their particular type of data/question.
    Formats Available: Streaming

    Approved Credit:
  • ISAC: 1.50 hours ICCE
  • ASCP: 1.50 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information Algorithmic Analysis of Flow Cytometry Data (Part 1) (2014 Scientific Tutorial)
    Collapse Algorithmic Analysis of Flow Cytometry Data (Part 2) (2014 Scientific Tutorial)
    This two-session tutorial will demonstrate the automated analysis of flow cytometry data using five different approaches. Traditional manual gating analysis suffers from poor reproducibility and objectivity, and is very difficult for high-dimensional data.  Several algorithms have now been developed, using different approaches that may be more suited to different datasets and biological questions.  Five approaches will be highlighted in this tutorial, covering a range of approaches from just identification of targeted or unknown populations, to a full pipeline of data analysis. The tutorials will consist of brief demonstrations of data analysis by each of the approaches. After the tutorials, the students should have an appreciation of the power of automated analysis, the requirements for entering their data into an automated analysis system, and how to choose the most appropriate analysis method for their particular type of data/question.
    Formats Available: Streaming

    Approved Credit:
  • ISAC: 1.50 hours ICCE
  • ASCP: 1.50 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information Algorithmic Analysis of Flow Cytometry Data (Part 2) (2014 Scientific Tutorial)
    Collapse Beyond Polychromatic: Spectral Flow Cytometry

    John P. Nolan, Ph.D. will be presenting the webinar "Beyond Polychromatic: Spectral Flow Cytometry."  John is the Past-President of ISAC and he is currently a Professor at the La Jolla Bioengineering Institute/The Scintillon Institute in San Diego, CA.

    Since the earliest days of flow cytometry, researchers have sought to bring the spectral resolution of a fluorescence spectrometer to the analysis of individual cells in flow, but faced many limitations in optics, detectors, and electronics that made this impractical. Today, these limitations have been largely overcome and it is possible to collect the full fluorescence emission spectra of individual cells in flow cytometers that use high efficiency prisms of gratings to disperse the light collected from single cells across a detector array. The ability to collect the full fluorescence spectrum allows compensation to be replaced by spectral unmixing, which can provide more accurate estimates of label intensity and resolve fluorochromes with significant spectral overlap.  Moreover, spectral flow cytometry opens the door to a new era of flow cytometry instruments and applications. Spectral flow cytometers have simpler optical paths and fewer components than conventional flow cytometers, potentially resulting in lower cost and maintenance. More importantly, spectral flow cytometry enables the use of new types of labeling probes. For example, nanoparticles exhibiting surface enhanced Raman scattering (SERS) have attracted interest for their multiplexing potential. In this webinar, I will describe the essential elements of spectral flow cytometry and show examples of fluorescence and SERS detection.

    Formats Available: Streaming, Webinar + Archive
    Original Seminar Date: June 19, 2014
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 1 hour ICCE
  • ASCP: 1 hour CMLE

  • Topics & Pricing InformationTopics & Pricing Information Beyond Polychromatic: Spectral Flow Cytometry
    Collapse Forensic Flow Cytometry

    Jennifer Wilshire will be presenting the webinar "Forensic Flow Cytometry." Jennifer is the Assistant Manager of the Flow Cytometry Core Facility at Memorial Sloan-Kettering Cancer Center in New York, NY.

    Do you sometimes look at flow cytometry results and wonder if something went wrong, but can't put your finger on it? In this webinar we will take a "forensic" approach to study the most common issues that give bad flow cytometry data.  We will use case studies and real examples to illustrate the causes of bad data and how you can prevent it. Many topics will be covered including staining issues, compensation, controls, and sorting. The focus of this webinar will be on understanding the potential pitfalls in flow cytometry experiments and learning practical methods to prevent these issues. After participating in this webinar, attendees will have practical suggestions of ways to ensure flow cytometry experiments will yield good data and purely sorted cells.

    Formats Available: Streaming
    Original Seminar Date: August 05, 2014
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 1 hour ICCE
  • ASCP: 1 hour CMLE

  • Topics & Pricing InformationTopics & Pricing Information Forensic Flow Cytometry
    Collapse Standardization of flow cytometry assays: What for? How? Where does it lead us?

    Tomáš Kalina, MD. PhD, is currently Associated Professor at Department of Pediatric Hematology and Oncology, Charles University in Prague, 2nd Faculty of Medicine, Czech Republic.

    He is a founding member of EuroFlow consortium where he is responsible for coordination of the technical aspects and design of flow cytometry procedures. At present, he is actively involved in EuroFlow Primary Immunodeficiency workpackage and EuroFlow proficiency testing. Tomáš is also an ISAC Scholar.

    Webinar will focus on standardization of the flow cytometry experimental procedures so that data analysis can be performed in an interlaboratory setting, on multiple instruments and over prolonged periods of time. Various aspects of reproducibility and robustness will be discussed. Since development of standardized flow cytometry tests is essential for meaningful computational analyses of the data, the new possibilities that are just opening will be discussed.

    Formats Available: Streaming
    Original Seminar Date: November 12, 2014
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 1 hour ICCE
  • ASCP: 1 hour CMLE

  • Topics & Pricing InformationTopics & Pricing Information Standardization of flow cytometry assays: What for? How? Where does it lead us?
    Collapse Predicting the best resolution and sensitivity in panel development and reducing inter-instrument variability in Flow Cytometry

    Presented by Steve Perfetto and Jim Wood. Moderated by Pratip Chattopadhyay

     Steve Perfetto

    Stephen P. received a B.S. degree in Medical Technology from the West Virginia University in 1977 and completed his M.S. degree in 1981 from West Virginia University. He studied and worked in the clinical Blood Bank and clinical immunology laboratories until 1988, when he joined the EPICS Division of Coulter Corporation. In 1990, he was recruited to the Walter Reed Army Institute of Research and was the manager of the core flow facility. While at WRAIR, he was involved in large HIV vaccine trials and developed functional to study the immune system of infected individuals.

    In 2000 he joined the NIH in the department of the Vaccine Research Center (VRC) as a staff scientist and manager of the flow core facility. This facility is the world-leader in multicolor flow cytometry and continues to actively develop this technology on a number of different fronts; one focus is on hardware development and reagent and analysis development. For several years, this lab has collaborated to develop Quantum Dots for use in immunophenotyping experiments. The advent of these fluorochromes provided an enormous advance in multicolor technology, allowing us to proceed from 12-color to 18-color very quickly. This group is actively working on new data analysis techniques - one major focus is the analysis and presentation of meta-data (for example, summarizing our functional analysis in which they have broken down each single response into hundreds of categories defined by the expression patterns of individual cytokines or other functional measurements). Recently, the Flow Cytometry Core broke the 18 color detection barrier by the discovery of 30 parameter flow cytometry and soon this will be further advanced to the world’s first 40 parameter instrument. Another focus is on particular aspects of immune function or viral dynamics, within the context of the major research efforts. (i) Detailed assays to an in vivo system to explore the role of CTL in lymphocyte dynamics during viral replication. (ii) Identification of viral reservoirs in CD4 T cell subsets. Sequence analysis of viruses isolated from specific CD4 T cell subsets give us an understanding of the spread of virus through the CD4 compartment, and the contribution of different CD4 subsets to both active viral production and latent reservoirs. (iii) Analysis and sorting of HIV Ag specific B cells to study neutralizing antibodies and to correlate the immunophenotyping of these cells using index sorting.

    James Wood

     

    James Wood obtained his BA in Physics from Gettysburg College; however, he also availed himself to a broad range of general and advanced biology, and chemistry courses during his undergraduate years.  He completed both his MS and PhD degrees in Biophysics from The Pennsylvania State University studying the effects on the life cycle of cells after radiation exposure.  In graduate school, he also developed the first microprocessor based two-parameter flow cytometer data acquisition system.  After completing a postgraduate position in the lab of Dr. Leon Wheeless at the University of Rochester Medical Center where he contributed to the development of a 3D slit-scan flow cytometer, he moved to Florida accepting a position with the EPICS Division of Coulter Corporation.  He was manager of the New Products Research and Applications Laboratory during most of his tenure at Coulter and Beckman-Coulter.  He currently consults for flow cytometry and pharmaceutical companies, and manages the flow cytometry shared resource at Wake Forest University School of Medicine Comprehensive Cancer Center (WFUCCC). 

    At the WFUCCC, he serves as a resource to the researchers and students.  He helps to design experiments and give advice on data interpretation.  He has worked to make flow cytometry an easily accessible technology for the Cancer Center users.  His knowledge of the technology as well as the associated biology of the applications enables him to guide the researchers and students in the successful application of the technology by explaining how to best probe the biological application with the flow cytometry technology.  He has a particular interest in the application of flow cytometry to the study of cell cycle kinetics and pursues this as time permits.  He enjoys sharing his passion for the flow cytometry technology and encouraging others to use it to its fullest extent.

    He is a long time member and contributor to International Society for the Advancement of Cytometry (ISAC) and, in the past, has served as chairperson and continues to be a member of the ISAC Data File Standards Task Force.  He has engaged in the research and development of the optical, fluidic, digital and analog electronics, and mechanical components of flow cytometers and cell sorters.  He has developed mathematical models to predict instrument performance, and has used the models to identify the critical features of a flow cytometer that limit the instrumentation’s performance. He has participated in the development of data analysis software for flow cytometry data and has worked on the development of mathematical models for cell life cycle analysis.  Currently, his interests have centered on (i) how to improve the instrument’s sensitivity and resolution of dim populations, (ii) optimizing the instrument’s ability to detect emissions from multiple fluorescent dyes, (iii) standardization of instrumentation setup and (iv) methods of data presentation and analysis. For these and other applications, he has been called upon as an expert resource to speak at users’ meetings and international courses.  He is actively developing standards for characterizing flow cytometer performance and is currently developing an LED based technology to optimize and standardize flow cytometry instrument setup.

    ---------------------------------

    Successful quantitative flow cytometry requires an understanding of the characteristics of a flow cytometer instrument that affect the measurement and analysis of the acquired photometric data.  Flow cytometer instruments must be characterized before being used for critical work.  The immediate goals include achieving better reproducibility of data, reducing variation and to facilitate intra/inter-lab comparisons of cytometry data.  Characterization  includes the determination of the instrument linearity, dynamic range, precision, accuracy, detection efficiency (Q), and electronic and optical noise (B).  In particular, Q and B affect how well cellular receptors with low expression can be measured.  For example, staining panels may be inadvertantly designed to avoid measuring dim markers on PMTs with poor resolution.  Unfortunately, current methods using bead sets to assess PMT resolution only provide rough estimates of Q and B.  Thus, staining panel design and similar protocols remain a largely empirical process, requiring time and intimate experience with an instrument’s performance. Ascertaining these instrument characteristics are the first steps toward assessing how instruments can be standardized and calibrated. Ultimately, this may be part of the validation process to certify that a flow cytometer used for critical clinical and/or GMP work is performing at the required level needed to obtain accurate and precise results.  Additionally, as part of this process we need to move from the common use of “relative intensity units (channel numbers)” to data measured and presented in physically relavent optical units (photons, photoelectrons, dye molecules, antibody binding sites).

    Recently, James Wood (Wake Forest University) developed a new device, known as an LED Pulser, to measure B and Q directly and accurately.  This device delivers consistent, uniform broad-spectrum light pulses, the amplitude of which can be adjusted with an electronic and/or an optical attenuator.  Our presentation will demonstrate how this device is used to calculate Q and B values.  In the past data from multilevel bead sets have been used to facilitate comparisons of instrument sensitivity with Q and B; however, it has proved difficult to manufacture bead sets that have closely matched intrinsic CVs.  In practice, the LED Pulser, along with calibrated fluorochrome-loaded beads, can used to determine the relationship between fluorescence channel numbers and the number of photoelectrons generated at the PMT photocathode i.e. the statistical photoelectron estimate (Spe) using a weighted quadratic fitting of pulsed LED series data.  This information, along with estimates of receptor density, can be used to calculate index values that provide quantitative guidance for panel design.  Our presentation will describe this process step-by-step, with examples.  Additionally, we will show how the metrics can be used for inter-laboratory comparisons.

    The first part of the webinar will present the principles behind the LED Pulser.  The appropriate use of the LED Pulser and how it compares to using multilevel bead sets for the calculation of Q and B. 

    The second part of the webinar will introduce how the LED Pulser, along with calibrated fluorochrome-loaded beads lead to better estimates of the four facets of predictive panel design (i) accurate Q and B values for each detector, (ii) optimal voltage settings, (iii) spillover/spreading error calculations, and (iv) estimates of receptor density and contribute to more quantitative determinations for optimizing panel design.

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    This continuing medical laboratory education activity is recognized by the American Society for Clinical Pathology for 1 CMLE credit.  ASCP CMLE credits are acceptable for the ASCP Board of Registry Certification Maintenance Program.

    Formats Available: Streaming
    Original Seminar Date: April 28, 2015
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 1 hour ICCE
  • ASCP: 1 hour CMLE

  • Topics & Pricing InformationTopics & Pricing Information Predicting the best resolution and sensitivity in panel development and reducing inter-instrument variability in Flow Cytometry
    Collapse Fluorescent Proteins in Flow Cytometry

    In a scientific tutorial in 2011, one of us (W.T.) covered the fluorescent proteins (FPs) available at the time. As a follow-up, we will focus on recent developments in the field, including new FPs, new lasers, and the advances made in detecting FPs using spectral flow cytometry. We will discuss considerations for practical use of FPs and make recommendations for choice of FPs in flow cytometry. We will analyze FP data using fluorescence compensation for conventional flow cytometry and unmixing algorithms for spectral flow cytometry. Attendees are encouraged to bring along their own data. Upon completion of this tutorial, the attendee will have an overview of the properties of commonly used FPs, the pertinent information to create optimal combinations of FPs, the knowledge to devise feasible detection strategies, and the skill to analyze FP data.

     

    Course Details or Outline:

    1. Basics/Overview Timeline of major developments in FPs List of commonly used FPs Considerations for practical use of FPs Recommendations for choice of FPs in flow cytometry
    2. Methods Strategies for simultaneous detection of multiple FPs Matching lasers with FPs Conventional vs spectral flow cytometry of FPs Flow cytometer calibration using FP beads
    3. Data Analysis Fluorescence compensation in conventional flow cytometry Unmixing algorithms in spectral flow cytometry Display of properly compensated FP data
    4. Discussion
    Formats Available: Streaming

    Approved Credit:
  • ISAC: 1.50 hours ICCE
  • ASCP: 1.50 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information Fluorescent Proteins in Flow Cytometry
    Collapse RNA Flow Cytometry

    Flow cytometry permits the simultaneous measurements of many biomarkers in individual cells from bulk populations. Until now analysis has been limited, however to primarily analysis of proteins and total DNA or highly abundant DNA sequences. Since most RNA gene transcripts are present at very low quantities our ability to detect these mRNA species by flow cytometry has been limited. In 1993, Patterson et. al. (Science, 1993. 260:976) used a PCR driven in situ hybridization technique to detect HIV mRNA in infected cells, however the stringent conditions required by this technique prevented its wide spread application to and development for flow cytometry. Two new techniques, PrimeFlowTM (Affymetrix/eBioscience) and SmartFlareTM (EMD Millipore) have recently been commercially introduced and are seeing increasing use among research scientists.

     

    This tutorial will discuss:

    • The methodology leading up to the current techniques
    • The PrimeFlowTM branched DNA methodology and applications
    • The SmartFlareTM technique and procedures
    • Pitfalls, quality control and analysis strategies for both Techniques
    • Methods to simultaneously combine mRNA measurements with labeling of surface and intracellular    proteins

     

    The PrimeFlowTM RNA assay system from Affymetrix/eBioscience allows for the determination of differential RNA expression within a mixed population of cells. The assay improves sensitivity and lowers background of fluorescent in situ hybridization (FISH) through the use of branched DNA signal amplification. Protocols and data will be presented showing the usefulness of this system to identify RNA expression on bone marrow B cells subsets. We will show a direct comparison of this method to a much more time consuming process of sorting cells for microarray analysis. This method provides a sophisticated molecular tool for the characterization of gene expression on rare cell subsets within tissue.

     

    The SmartFlareTM system from EMD Millipore employs a gold nanoparticle that is actively endocytosed into most cell types. In the cytoplasm, interaction with the specific mRNA target causes the generation of a fluorescent signal, which can be detected by flow cytometry or microscopy. Protocols and data will be presented showing the usefulness of this system to sort cells based on mRNA expression, for several applications such as isolation of cancer stem cells from tumors and optimizing shRNA or CRISPR knockdown clone selection. This new technology now enables the non-destructive detection of intracellular epitopes in living cells.

     

    By the end of this tutorial, attendees will have an understanding of which technique is most appropriate for their applications; how to apply them to their research and most importantly gain an understanding of the pitfalls and solutions from individuals who have learned these the hard way.

     

    Course Details or Outline:

     A. Introduction

    1. Overview of methods to detect RNA in bulk preparations of cells

    2. Initial flow cytometric methods to detect HIV by in situ PCR

    3. RNA-Seq and Fluidigm methodogies

    B. PrimeFlow - branched DNA

    1. Theory

    2. Method and data analysis

    3. Pitfalls

    4. Comparison of flow to microarray based expression profiling

    C. SmartFlare

    1. Theory of detecting RNA in live cells

    2. Protocols and data analysis

    3. Pitfalls

    4. Use for isolation of cancer stem cells from tumors

    5. Optimizing shRNA or CRISPR knockdown clone selection

     

    Formats Available: Streaming

    Approved Credit:
  • ASCP: 1.50 hours CMLE
  • ISAC: 1.50 hours ICCE

  • Topics & Pricing InformationTopics & Pricing Information RNA Flow Cytometry
    Collapse RNA Flow Cytometry by Steven McClellan and Paul Wallace

    Steve McClellanSteven McClellan, Flow Cytometry Core Laboratory, Mitchell Cancer Institute, University of South Alabama, Mobile, AL.

    Steve McClellan, BS, MT(ASCP) is a life science professional with over 25 years of expertise in advanced flow cytometry & cell sorting, as well as basic and clinical research in the areas of cancer biology, stem cell therapy, transplant immunology and xenotransplantation. He is currently Manager of Basic & Translational Research Operations at the University of South Alabama Mitchell Cancer Institute, where he also serves as Chief, Flow Cytometry & Imaging Core Laboratories. For the past seven years, his lab has been conducting research on cancer stem cells; working to develop better methods of purification, culture and analysis at both genetic and functional levels, as well as using cancer stem cells in HTS drug discovery.

     

    Paul WallacePaul K. Wallace, Department of Flow & Image Cytometry, Roswell Park Cancer Institute, Buffalo, NY

    Paul K. Wallace, PhD has served since 2003 as Director of the Flow and Image Cytometry Department and is Professor of Oncology at Roswell Park Cancer Institute (RPCI) in Buffalo, NY. He is also Associate Professor of Microbiology & Immunology, Dartmouth College, Hanover, NH and Associate Professor of Biotechnical and Clinical Laboratory Sciences, University at Buffalo, Buffalo, NY. He is President elect of the International Society for Advancement of Cytometry and a Councilor of the International Clinical Cytometry Society.

    Under his direction, the Flow and Image Cytometry Department at RPCI offers a strong combination of clinical and research missions.  The department’s clinical emphasis is on the diagnosis and monitoring of patients with leukemia and lymphoma.  In addition, it serves as a core reference facility performing immunophenotyping and immune monitoring studies on samples from patients enrolled in clinical trials for several biotech and grant-funded organizations.  The department’s research focus is on myeloid cell biology and translational research utilizing flow cytometry.

    Before joining RPCI, Dr. Wallace was an Assistant Professor of Immunology at Dartmouth Medical School, Hanover NH (1993-2003), a cofounder of Zynaxis Cell Science, Inc., Malvern PA (1988-1991), and the Supervisor of Flow Cytometry at SmithKline (now Quest) Clinical Laboratories King of Prussia, PA (SKCL; 1979-1988).  He is internationally recognized for his commitment to flow cytometric education and has been a member of ISAC’s Educational Task Force/Committee since its inception in 2006 and of the ICCS Education committee since 2003. He is a consultant with ASCP and CDC’s PETFAR (U.S. President's Emergency Plan for AIDS Relief), for which he has developed and presented CD4 training programs in Nigeria, India, Mozambique and Vietnam. Since 1994 he has also been on the faculty of the Bowdoin/New Mexico Annual Course in Methods and Applications of Cytometry.

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    Webinar Summary: 

    Flow cytometry permits the simultaneous measurements of many biomarkers in individual cells from bulk populations. Until now analysis has been limited, however to primarily analysis of proteins and total DNA or highly abundant DNA sequences. Since most RNA gene transcripts are present at very low quantities our ability to detect these mRNA species by flow cytometry has been limited. In 1993, Patterson et. al. (Science, 1993. 260:976) used a PCR driven in situ hybridization technique to detect HIV mRNA in infected cells, however the stringent conditions required by this technique prevented its wide spread application to and development for flow cytometry. Two new techniques, PrimeFlowTM (Affymetrix/eBioscience) and SmartFlareTM (EMD Millipore) have recently been commercially introduced and are seeing increasing use among research scientists.

    The PrimeFlow RNA assay system allows for the determination of differential RNA expression within a mixed population of cells. The assay improves sensitivity and lowers background of fluorescent in situ hybridization (FISH) through the use of a branched DNA signal amplification. In contrast, the SmartFlareTM system employs a gold nanoparticle that is actively endocytosed into most cell types. In the cytoplasm, interaction with the specific mRNA target causes the generation of a fluorescent signal, which can be detected by flow cytometry or microscopy. Protocols and data will be presented showing the usefulness of both systems. By the end of the webinar, participants should have an understanding of which technique is most appropriate for their applications; how to apply them to their research and most importantly gain an understanding of the pitfalls and solutions from individuals who have learned these the hard way.

    This continuing medical laboratory education activity is recognized by the American Society for Clinical Pathology for 1 CMLE credit. ASCP CMLE credits are acceptable for the ASCP Board of Registry Certification Maintenance Program.

    Formats Available: Streaming
    Original Seminar Date: August 26, 2015
    On-Demand Release Date: Available Now

    Approved Credit:
  • ASCP: 1 hour CMLE
  • ISAC: 1 hour ICCE

  • Topics & Pricing InformationTopics & Pricing Information RNA Flow Cytometry by Steven McClellan and Paul Wallace
    Collapse Proliferation Monitoring-Lesson 1: Basics of the Cell Cycle

    Authors: Paul K. Wallace, Kathy Muirhead and Kylie Price

    Course Objectives:

    • To understand what cellular processes need to occur in order for a cell to enter the cell cycle and complete division.
    • To know and understand the difference between cell cycle progression and cell division
    • To understand how flow cytometry is used to measure cell cycle parameters and division

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    To view the course, select "Topics & Pricing Information" and then select "Add to Basket."

    If you are not logged in to the CYTO U website, you will be directed to a new page asking for your login information.  
     
    For current ISAC members, your user name and password to CYTO U are the same user name and password you have for the members-only section of the ISAC Web site. If you cannot remember your password, you can re-set it on the ISAC Web site by clicking on the "Forgot Password?" link on the log-in page.
     
    --------------
    This continuing medical laboratory education activity is recognized by the American Society for Clinical Pathology for 1 CMLE credit.  ASCP CMLE credits are acceptable for the ASCP Board of Registry Certification Maintenance Program.
     
    Formats Available: Streaming
    Original Seminar Date: September 02, 2015
    On-Demand Release Date: Available Now

    Approved Credit:
  • ASCP: 1 hour CMLE
  • ISAC: 1 hour ICCE

  • Topics & Pricing InformationTopics & Pricing Information Proliferation Monitoring-Lesson 1: Basics of the Cell Cycle
    Collapse Flow Cytometric Analysis of Endothelial Colony Forming Cells and Hematopoietic Progenitor Cells in Lung Vascular Disease by Kewal Asosingh and Imaging Flow Cytometry in the Study of Immune Cell Functions by Andrew Filby

    Kewal Asosingh

    Flow Cytometric Analysis of Endothelial Colony Forming Cells and Hematopoietic Progenitor Cells in Lung Vascular Disease presented by Kewal Asosingh (ISAC Scholar)

    Dr. Asosingh is a Staff Scientist, Assistant Professor of Molecular Medicine and Scientific Director for Flow Cytometry at Cleveland Clinic’s Lerner Research Institute. In 2011, he was chosen by the International Society for Advancement of Cytometry (ISAC) as an ISAC Scholar, recognition of emerging scientific research leaders in the profession. He has provided Stem Cell Cytometry training seminars at ISAC meetings, chaired oral presentation sessions on personalized medicine, and has been serving as abstract reviewer, member of the program committee for the annual ISAC meetings (“CYTO”) and reviewer for Cytometry Part A. Dr. Asosingh is also co-leader of the CYTO University (CYTO U) eLearning Delivery Task Force. Dr. Asosingh teaches flow cytometry to undergraduate and graduate students at the Cleveland Clinic and at Lakeland Community College. As Scientific Director of the Flow Cytometry Core, he serves as primary contact with investigators to provide advice on experimental design and data analysis/interpretation and assures that data generated by the Flow Core is of the highest quality.

    Webinar Summary:

    Dr. Asosingh’s lab studies bone marrow stem cells in pulmonary arterial hypertension (PAH) and pathological angiogenesis in asthma. The group has established mouse models for both diseases, and close collaboration with clinicians facilitates rapid transition of the basic research findings to clinical studies. In the past decade it has become evident that bone marrow hematopoietic stem cells exert functions beyond hematopoiesis. During development, endothelial cells and hematopoietic stem cells originate from a common bipotent mesodermal stem cell called the hemangioblast. In post-natal life, this connection is maintained via paracrine interactions between the endothelium and circulating hematopoietic progenitor cells. Their critical role in vascular homeostasis and repair of endothelial injury has opened new therapeutic perspectives for vascular degenerative diseases. The flipside of the coin is that abnormalities in hematopoiesis may underlie pathological angiogenesis, and this is exactly what his group is focused on.  Recent findings from the group show that hematopoietic stem cells isolated from the bone marrow of PAH patients are able to transfer the disease in humanized NOD SCID mice. PAH is a devastating disease characterized by endothelial cell injury, in situ thrombi and right ventricular hypertrophy. Animals engrafted with hematopoietic stem cells from PAH patients, but not from healthy control participants, had increased mobilization of progenitor cells in the peripheral blood, just like in patients, and strikingly developed many features of PAH.  Current focus of the team is to further reveal the mechanisms by which hematopoietic stem cells cause pulmonary vascular disease. This webinar will provide an overview of the different types of “endothelial progenitor cells” with special emphasis on endothelial colony forming cells and pro-angiogenic hematopoietic progenitor cells and their roles in PAH.

    Andrew Filby

    Imaging Flow Cytometry in the Study of Immune Cell Functions presented by Andrew Filby (SRL Emerging Leader)

    Dr Filby is currently head of the Flow Cytometry Core Facility at Newcastle University.  He leads a dedicated team of flow cytometry specialists with the sole aim of providing a comprehensive, cutting edge cytometry resource to the wider research community at Newcastle University and beyond.  A significant part of his focus is the development of novel cytometry-based techniques that have underpinned several high profile publications including in Science, Cell and the Cytometry Part A paper of the year accolade (2011).  He specialises in Imaging Flow Cytometry and the use of fluorescence dyes to track cell proliferation.  Prior to and post selection as an ISAC Shared Resource Laboratory Emerging Leader (SRLEL), Dr Filby has made several contributions to the Cyto conference programmes including interactive workshops and scientific talks.  He continues to contribute to the efforts of ISAC including serving on the Image Cytometry and E-learning task forces.  In addition to his work within ISAC, he is also heavily involved in supporting cytometry research and education in South America including initiatives for remote support and training.

    Webinar Summary:

    In this short webinar Dr Filby will give a brief overview of his work within the field of Imaging Flow Cytometry and what impact this has had on a number of key publications in the field of immunology and cell cycle biology.  These include the study of asymmetric cell division in the immune system, the measurement of organelle-specific calcium mobilisation in activated T cells and the analysis of the cell cycle in fission yeast.  In each case, the ability to capture and analyse literally thousands of multispectral, spatially registered images was essential to questions each method was designed to address.  Finally, Dr Filby will also discuss what the SRL EL programme means to him and how he hopes it will benefit the wider cytometry community through various initiatives he is involved in.

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    This continuing medical laboratory education activity is recognized by the American Society for Clinical Pathology for 1 CMLE credit. ASCP CMLE credits are acceptable for the ASCP Board of Registry Certification Maintenance Program.

    Formats Available: Streaming, Webinar + Archive
    Original Seminar Date: November 16, 2015
    On-Demand Release Date: Available Now

    Approved Credit:
  • ASCP: 1 hour CMLE
  • ISAC: 1 hour ICCE

  • Topics & Pricing InformationTopics & Pricing Information Flow Cytometric Analysis of Endothelial Colony Forming Cells and Hematopoietic Progenitor Cells in Lung Vascular Disease by Kewal Asosingh and Imaging Flow Cytometry in the Study of Immune Cell Functions by Andrew Filby
    Collapse Small Instruments for Large Tasks:  High Dimensional Flow Cytometry on Compact Platforms by Bill Telford. Moderated by Paul Wallace.

    Bill Telford  Bill Telford

    • Ph.D. Michigan State University (1994)

    • Postdoc, University of Michigan (1994-1997)

    • Flow Core Director, Hospital for Special Surgery/Cornell University School of Medicine (1997-1999)

    • Flow Facility Manager and Associate Scientist, National Cancer Institute, NIH (1999-present)

    Webinar Summary:

    Modern optical and electronic technology are allowing the development of small, compact flow cytometers with the capabilities of much larger instrumentation.  We will discuss several of these cytometer platforms, and the technological advances that have made them possible.

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    This continuing medical laboratory education activity is recognized by the American Society for Clinical Pathology for 1 CMLE credit.  ASCP CMLE credits are acceptable for the ASCP Board of Registry Certification Maintenance Program.

     

    Formats Available: Streaming, Webinar + Archive
    Original Seminar Date: April 13, 2016
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 1 hour ICCE
  • ASCP: 1 hour CMLE

  • Topics & Pricing InformationTopics & Pricing Information Small Instruments for Large Tasks:  High Dimensional Flow Cytometry on Compact Platforms by Bill Telford. Moderated by Paul Wallace.
    Collapse CYTO 2013: Advanced Data Analysis
    Data analysis is a critical, complex component of flow cytometry experiments. In this course, we demonstrate how data analysis is relevant in nearly every experimental phase. The course begins with a module on experimental design and data acquisition, in which instrument standardization, panel design, and proper controls will be discussed from a data analysis perspective. Next, a module on data visualization will cover population identification (e.g., "gating"), data analysis in the cloud, and troubleshooting based on staining patterns. Following this module, during the lunch break, vendors will be available to demonstrate software packages and answer questions. The afternoon includes sessions on reporting results (fundamental statistics, data aggregation for presentation) and the next generation of R-based tools for automated data analysis. A course packet will be provided with supplementary materials, including review articles and cytometry list discussions that address frequently asked (and complex) questions, like "how many events should be collected?" or "what is the value of isotype controls?" In sum, this course takes a fresh look at the fundamentals of data analysis and introduces cutting-edge tools for the future.

    All CYTO U courses qualify for ICCE continuing education credit. If you would like to see a list of topics included in this recording, please click on the "Topics" tab.
    Formats Available: Streaming
    Original Seminar Date: May 18, 2013
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 5.50 hours ICCE
  • ASCP: 5.50 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information CYTO 2013: Advanced Data Analysis
    Collapse Preventing GI/GO Syndrome Instrument Standardization (2013 Advanced Data Analysis PreCongress Course)
    Data analysis is a critical, complex component of flow cytometry experiments. In this course, we demonstrate how data analysis is relevant in nearly every experimental phase. The course begins with a module on experimental design and data acquisition, in which instrument standardization, panel design, and proper controls will be discussed from a data analysis perspective. Next, a module on data visualization will cover population identification (e.g., "gating"), data analysis in the cloud, and troubleshooting based on staining patterns. Following this module, during the lunch break, vendors will be available to demonstrate software packages and answer questions. The afternoon includes sessions on reporting results (fundamental statistics, data aggregation for presentation) and the next generation of R-based tools for automated data analysis. ;A course packet will be provided with supplementary materials, including review articles and cytometry list discussions that address frequently asked (and complex) questions, like "how many events should be collected?" or "what is the value of isotype controls?" In sum, this course takes a fresh look at the fundamentals of data analysis and introduces cutting-edge tools for the future.

    Topics covered in this session include:
    • How to Prevent GI/GO (Garbage In/Garbage Out) Syndrome Instrument Standardization
    • Panel design
    All CYTO U courses qualify for ICCE continuing education credit.
    Formats Available: Streaming
    Original Seminar Date: May 18, 2013
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 0.50 hours ICCE
  • ASCP: 0.50 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information Preventing GI/GO Syndrome Instrument Standardization (2013 Advanced Data Analysis PreCongress Course)
    Collapse Flow Cytometry Controls (2013 Advanced Data Analysis PreCongress Course)
    Data analysis is a critical, complex component of flow cytometry experiments. In this course, we demonstrate how data analysis is relevant in nearly every experimental phase. The course begins with a module on experimental design and data acquisition, in which instrument standardization, panel design, and proper controls will be discussed from a data analysis perspective. Next, a module on data visualization will cover population identification (e.g., "gating"), data analysis in the cloud, and troubleshooting based on staining patterns. Following this module, during the lunch break, vendors will be available to demonstrate software packages and answer questions. The afternoon includes sessions on reporting results (fundamental statistics, data aggregation for presentation) and the next generation of R-based tools for automated data analysis. A course packet will be provided with supplementary materials, including review articles and cytometry list discussions that address frequently asked (and complex) questions, like "how many events should be collected?" or "what is the value of isotype controls?" In sum, this course takes a fresh look at the fundamentals of data analysis and introduces cutting-edge tools for the future.

    Topics covered in this session include:

  • Flow Cytometry Controls

  • All CYTO U courses qualify for ICCE continuing education credit.
    Formats Available: Streaming
    Original Seminar Date: May 18, 2013
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 0.50 hours ICCE
  • ASCP: 0.50 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information Flow Cytometry Controls (2013 Advanced Data Analysis PreCongress Course)
    Collapse Visualizing Data and Identifying Populations (2013 Advanced Data Analysis PreCongress Course)
    Data analysis is a critical, complex component of flow cytometry experiments. In this course, we demonstrate how data analysis is relevant in nearly every experimental phase. The course begins with a module on experimental design and data acquisition, in which instrument standardization, panel design, and proper controls will be discussed from a data analysis perspective. Next, a module on data visualization will cover population identification (e.g., "gating"), data analysis in the cloud, and troubleshooting based on staining patterns. Following this module, during the lunch break, vendors will be available to demonstrate software packages and answer questions. The afternoon includes sessions on reporting results (fundamental statistics, data aggregation for presentation) and the next generation of R-based tools for automated data analysis. A course packet will be provided with supplementary materials, including review articles and cytometry list discussions that address frequently asked (and complex) questions, like "how many events should be collected?" or "what is the value of isotype controls?" In sum, this course takes a fresh look at the fundamentals of data analysis and introduces cutting-edge tools for the future.

    Topics covered in this session include:
    • Visualizing Data
    • Identifying Populations

    All CYTO U courses qualify for ICCE continuing education credit.
    Formats Available: Streaming
    Original Seminar Date: May 18, 2013
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 0.50 hours ICCE
  • ASCP: 0.50 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information Visualizing Data and Identifying Populations (2013 Advanced Data Analysis PreCongress Course)
    Collapse Mathematical Modeling for Cytometry (2013 Advanced Data Analysis PreCongress Course)
    Data analysis is a critical, complex component of flow cytometry experiments. In this course, we demonstrate how data analysis is relevant in nearly every experimental phase. The course begins with a module on experimental design and data acquisition, in which instrument standardization, panel design, and proper controls will be discussed from a data analysis perspective. Next, a module on data visualization will cover population identification (e.g., "gating"), data analysis in the cloud, and troubleshooting based on staining patterns. Following this module, during the lunch break, vendors will be available to demonstrate software packages and answer questions. The afternoon includes sessions on reporting results (fundamental statistics, data aggregation for presentation) and the next generation of R-based tools for automated data analysis. ;A course packet will be provided with supplementary materials, including review articles and cytometry list discussions that address frequently asked (and complex) questions, like "how many events should be collected?" or "what is the value of isotype controls?" In sum, this course takes a fresh look at the fundamentals of data analysis and introduces cutting-edge tools for the future.

    Topics covered in this session include:
    • Mathematical Modeling

    All CYTO U courses qualify for ICCE continuing education credit.
    Formats Available: Streaming
    Original Seminar Date: May 18, 2013
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 1 hour ICCE
  • ASCP: 1 hour CMLE

  • Topics & Pricing InformationTopics & Pricing Information Mathematical Modeling for Cytometry (2013 Advanced Data Analysis PreCongress Course)
    Collapse SPADE (2013 Advanced Data Analysis PreCongress Course)
    All CYTO U courses qualify for ICCE continuing education credit.
    Formats Available: Streaming
    Original Seminar Date: May 18, 2013
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 1 hour ICCE
  • ASCP: 1 hour CMLE

  • Topics & Pricing InformationTopics & Pricing Information SPADE (2013 Advanced Data Analysis PreCongress Course)
    Collapse CYTO 2013: Scientific Tutorials
    All CYTO U courses qualify for ICCE continuing education credit. If you would like to see a list of topics included in this recording, please click on the "Topics" tab.
    Formats Available: Streaming
    Original Seminar Date: May 18, 2013
    On-Demand Release Date: Available Now

    Approved Credit:
  • ISAC: 15.00 hours ICCE
  • ASCP: 15.00 hours CMLE

  • Topics & Pricing InformationTopics & Pricing Information CYTO 2013: Scientific Tutorials