About the Speaker

Dr. Kewal Asosingh
Scientific Director for Flow Cytometry
Cleveland Clinic Lerner Research Institute

Dr. Asosingh is a principal investigator and scientific director for flow cytometry at the Cleveland Clinic Lerner Research Institute. He is a past ISAC Scholar, Cytometry Part A associate editor, ISAC Flow Cytometry Content subcommittee chair, and member of the Education Committee. He has more than 20 years of experience in solid tissue disaggregation into a single-cell suspension.

Webinar Summary

A good single-cell prep is essential for any flow cytometry and single-cell omics experiment. This webinar outlines the principles and provides a general guide of steps to consider for solid tissue disaggregation.

Learning Objectives:

• Basics of solid tissue composition.
• What is a “good” single-cell prep in flow cytometry and single-cell RNAseq experiments?
• The dos and don’ts in designing a tissue disaggregation protocol.
• Evaluation of the quality of a single cell prep and common pitfalls. 

Who Should Attend

Flow cytometry SRL staff and anyone performing flow cytometry using solid tissue as starting material. 

CMLE Credit: 1.0

About the Speakers

Sherry Thornton, PhD
Professor of Rheumatology
Director, Research Flow Cytometry Core

Dr. Sherry Thornton been involved in flow cytometry for over 19 years both in projects as an investigator and in provision of services as a core director. She is a field service professor in the Department of Pediatrics at the University of Cincinnati College of Medicine (UCCOM) in the Division of Rheumatology at Cincinnati Children’s Hospital Medical Center (CCHMC). Her main role is to direct the research flow cytometry core and provide services to over 190 investigators and their labs both at CCHMC and UCCOM. She is highly involved in education and shared facilities nationally and internationally. She currently serves as chair of the Education Committee of the International Society for the Advancement of Cytometry (ISAC) and is a past chair and current member of the Flow Cytometry Research Group for Association for Biomedical Resource Facilities (ABRF). She also was involved in the launching and the continuation of the ABRF mentorship program as a member of the Career Development Committee. In striving to provide education and state-of-the-art flow cytometry for core users, she has been supported by two NIH P30 grant mechanisms for over 15 years and has been awarded two NIH Shared Instrumentation Grants to provide access to user-friendly based cell sorting 24/7 and high parameter flow cytometry. 

Julia Fernandez-Rodriguez, PhD
Sahlgrenska Academy
University of Gothenburg, Sweden

Julia's research career in cell and molecular biology and core facility training experience has provided her with an excellent background in multiple life sciences disciplines, as well as in the management, operation, and coordination of an imaging research infrastructure and its training activities at the national and international level. Since 2003 Julia has been responsible of the Centre for Cellular Imaging, an open-access Correlated Multimodal Imaging Facility that provides technical and scientific excellence by integrating multiple imaging technologies with image processing and analysis tools in a single core. Julia's main interest is to provide expertise in correlated multimodal imaging workflows (from experimental design to image acquisition and analysis) tailored to various research domains within the life sciences. In 2016, Julia was awarded one of the 15 Research Infrastructure Fellows grants by the Swedish Foundation for Strategic Research. At the university core facility, Julia is also involved in the education and training of students and researchers through a series of courses, seminars, and workshops, often in collaboration with other universities in Sweden and abroad and with industrial partners. Julia's responsibility is to organize and lead these events and ensure that the scientific community receives the appropriate basic or advanced training on different microscopy methods. Julia's overall aim is to have students and researchers foster a deep understanding of basic and advanced methods used in microscopy to tackle their questions about the most appropriate probes and instruments. Furthermore, she has supervised core internship students in bioimaging, promoting future career possibilities and introducing core facility work as a possible career direction. At the national level, she is the scientific coordinator of the National Microscopy Infrastructure in Sweden and a member of the Boards of the Nordic Microscopy Society (SCANDEM) and the Bridging Nordic Microscopy Infrastructure (NordForks). Julia is also connected to several other European facilities such as the European Light Microscopy Initiative, ELMI (member of the Steering committee since 2008), and the Euro-BioImaging ERIC consortium (member of the Nodes Board). She is also president of the Core Technologies for Life Sciences Association (CTLS). Further, she represent Sweden in the Management Committee and is the coordinator of the Short-Term Scientific Missions of the European COST Action COMULIS (CA17121) -funded network in Correlated Multimodal Imaging in Life Sciences.

Webinar Summary

Participants are invited to learn tips and tricks regarding funding for shared resource laboratories (SRL). Speakers will address NIH and European funding sources, focusing on how participants can support their cytometry SRL through instrumentation or other grant mechanisms.

Learning Objectives

• Identify funding sources for shared resource laboratories.
• Determine basic requirements for successful core laboratory grant submissions.
• Provide specific tips for successful grant submissions.
• Determine common pitfalls for grant applications.

CMLE Credit: 1.0

About the Presenter

Thomas Liechti, PhD
Postdoctoral Researcher
ImmunoTechnology Section
Vaccine Research Center
National Institutes of Health

Thomas Liechti obtained his PhD in immunology and microbiology at the University of Zurich in 2017 and is currently a postdoctoral researcher in Mario Roederer’s group at the Vaccine Research Center of the National Institutes of Health in Bethesda (USA). His main interest is high-dimensional flow cytometry and human immunology. During his postdoctoral training, he established a 28-color flow cytometry sample processing and analysis pipeline to assess the contribution of genetic and environmental factors to human immune homeostasis in a cohort of more than 3000 individuals.  

Florian Mair, PhD
Research Associate | Cytometry Specialist
Fred Hutchinson Cancer Research Center

Florian Mair graduated with a PhD from the University of Zurich, Switzerland, in 2014 and is currently working at the Fred Hutchinson Cancer Research Center in Seattle, USA, as a research associate. During the past decade, he has been involved extensively with different cytometry platforms (conventional, spectral and mass cytometry) as well as scRNA-seq techniques and developed an interest in applying novel analysis approaches for single cell data. He has been actively engaged in teaching flow cytometry courses, including systematic panel design and analysis of high-dimensional cytometry experiments. 

Webinar Summary

Human dendritic cells (DCs) and monocytes are critical components of the innate immune system and important for orchestrating adaptive immunity. In recent years high-dimensional single cell technologies, such as flow cytometry and single cell RNA-seq, dissected the heterogeneity of human DCs and monocytes more precisely, suggesting more robust markers to unequivocally define these subsets. Based on these results and our recently published phenotype report (Mair F. and Liechti T. Cytometry Part A, 2021), we will discuss strategies to better define and characterize human DCs and monocytes using a combination of traditional and recently discovered markers. We will show practical examples of how the combination of thorough panel design and unsupervised data analysis can help to dissect heterogenous immune populations. This webinar will be split into three parts:

• A description about the phenotypic and functional characteristics of human DC and monocyte subsets with a focus on how the emergence of single-cell technologies improved our understanding of the heterogenous landscape of human phagocytes.
• High-dimensional panel design for characterization of human DCs and monocytes with a more specific focus on marker selection and the incorporation of biological knowledge into this process.
• An overview of how unsupervised data analysis approaches can improve the delineation of human DC and monocyte subsets.

Learning Objectives

• Learn about the phenotypic and functional characteristics of human DCs and monocytes.
• Understand strategies for marker selection in high-dimensional flow cytometry panel design.
• Learn how unsupervised data analysis can guide the analysis of human DCs and monocytes.

 Who Should Attend

Scientist interested in studying human dendritic cells and monocytes using flow cytometry. In addition, flow cytometrists with a general interest in high-dimensional flow cytometry panel design.

CMLE Credit: 1.0

About the Speaker

Jakob Zimmerman, PhD
University of Bern
Department for BioMedical Research

Dr. Zimmermann obtained his PhD in the lab of former ISAC president Andreas Radbruch at the German Rheumatism Research Center in Berlin, Germany, working on Th cells in the pathogenesis of IBD, while also establishing new methods for the flow cytometric interrogation of the intestinal microbiota. As a postdoctoral fellow supported by a Marie-Curie fellowship by the European Commission, he moved to the lab of Andrew Macpherson at the University of Bern, Switzerland, to further specialize in host-microbe mutualism. As an ISAC Marylou-Ingram scholar, he is currently leveraging the power of bacterial flow cytometry when combined with the robustness of defined gnotobiotic model microbiotas.

Webinar Summary

The intestinal microbiota has been implicated in nearly all aspects of human health, yet our mechanistic understanding of these microbial consortia and their interaction with the host remains superficial. This webinar shall address how flow cytometry and cell sorting of gut bacteria can contribute to deepen this knowledge. It’ll involve key advantages over other techniques, examples for its application as well as practical guidance and pitfalls when doing microbiota flow cytometry.

Learning Objectives

Attendants should learn how flow cytometry can contribute to research on the intestinal microbiota and which questions can best be answered using microbiota flow. Topics that will be discussed include how gnotobiotic mice with defined microbiotas can be leveraged for robust microbiome research and why they are particularly powerful when combined with microbiota flow cytometry and cell sorting. A key learning objective is also how to do microbiota flow cytometry and sorting as well as important controls and potential pitfalls.

Who Should Attend

As the gut microbiota affects almost all human (and mouse…) organ systems, the webinar is targeted at immunologists and cancer researchers just as much as at microbiologists and microbiota scientists.

CMLE Credit: 1.0

About the Presenter

Jessica B. Back, PhD, SCYM(ASCP)CM
Deputy Director
Microscopy, Imaging, and Cytometry Resources Core
Karmanos Cancer Institute, Wayne State University

Jessica B. Back, Ph.D., SCYM(ASCP)^CM is a research scientist and deputy director of the Microscopy, Imaging, and Cytometry Resources (MICR) Core at the Karmanos Cancer Institute and Wayne State University in Detroit, MI. She received her PhD in biochemistry from Wayne State University and completed her post-doctoral training in tumor immunology at the Karmanos Cancer Institute. Jessica is an ISAC councilor, a former ISAC SRL emerging leader (2015-2019), a member of the Great Lakes International Imaging and Cytometry Association (GLIIFCA) Board of Directors, and chair of the American Society of Clinical Pathologists (ASCP) Board of Certification Cytometry Examination Committee. As Deputy Director of an SRL, much of her focus is on efficient and effective delivery of services to facility users. Her research interests focus on the tumor microenvironment, particularly on the role the immune system plays in cancer therapies and tumor regression.

Ann Marie DesLaurieres-Cox
Assistant Manager, Flow Cytometry Core
University of Michigan

Ann Marie DesLauriers-Cox is the assistant manager of the Flow Cytometry Core Facility (FCCF) at the University of Michigan in Ann Arbor, Michigan. She received her BS in clinical lab sciences at Eastern Michigan University. Ann Marie has been a technician in the Flow Core for 25 years, where she focuses on customer relations, training, maintenance, and troubleshooting of the Flow Cytometers. She has presented previous “soft skills” workshops at both CYTO and GLIIFCA conferences.

Webinar Summary

Shared Resources Laboratory (SRL) personnel often assume a role as scientific mentor to trainees within their institutions. In this position, they may see and hear things from these trainees that fly under the radar of their mentors and occasionally result in an overflow of emotions in the SRL facility. As such, SRL staff need to be equipped with emotional intelligence to navigate these interactions, provide support to users if needed, and alert the mentor or administration if required, all while maintaining a professional working environment. The goal of this CYTO U session is to provide a brief introduction to emotional intelligence and a framework SRL personnel may use to navigate these interactions with users and colleagues.

Learning Objectives

• Familiarize the audience with the concept of emotional intelligence.
• Introduce a framework for the practical application of emotional intelligence within the SRL.

Who Should Attend

• RL Managers and Personnel.

CMLE Credit: 1.0

About the Presenter

Anna Belkina, MD, PhD
Assistant Professor of Pathology and Laboratory Medicine
Director, Flow Cytometry Core Facility
Boston University School of Medicine

Anna C. Belkina is an assistant professor of pathology and laboratory medicine and the director of the Flow Cytometry Core Facility at Boston University School of Medicine. She received her MD degree from Russian State Medical University in Moscow and her PhD degree from Boston University School of Medicine investigating the epigenetic regulation of inflammatory responses driven by bromodomain proteins. Anna’s research is focused on the intersection of immunology and computational biology and her research efforts include investigating the immune landscape of chronic inflammatory diseases and developing computational techniques to assess high-parameter single cell cytometry data. She has designed the opt-SNE algorithm that is now widely used for the visualization of multidimensional cytometry datasets. 
Anna is an active member of ISAC and has been named a 2015–2019 ISAC SRL Emerging Leader. She is a member of the ISAC Council elected for the 2020–2024 term.

Webinar Summary

Visualization of multiparameter datasets is a staple task in the data analysis pipeline. Over last few years, multiple dimensionality reduction algorithms have been adopted for visual presentation of cytometry data to aid identification of novel cell populations and biological trends. In this webinar, we will discuss the basic principles of these approaches and identify benefits and drawbacks of several mainstream algorithms including popular variants of t-SNE and UMAP. We will specifically highlight the importance of hyperparameter optimization for cytometry datasets and practical considerations of choosing the suitable computational environment for your analysis. 

Learning Objectives

• Familiarize the audience with recent advances in dimensionality reduction approaches in cytometry datasets. 
• Introduce and discuss the basic principles of popular dimensionality reduction algorithms and compare their strengths and weaknesses.
• Demonstrate how computational analysis enhances the power of high parameter flow cytometry.

Who Should Attend

Cytometry practitioners and data analysts who encounter multidimensional datasets and employ algorithmic data analysis approaches or are interested in learning these methods

CMLE Credit: 1.0

Speaker

Sharon Lewin, AO, FRACP, FAHMS
Director, Peter Doherty Institute for Infection and Immunity

Session Summary

Leading infectious diseases expert, Professor Sharon Lewin, is the inaugural Director of the Doherty Institute. She is also a Professor of Medicine at The University of Melbourne and a National Health and Medical Research Council (NHMRC) Practitioner Fellow. As an infectious diseases physician and basic scientist, her laboratory focuses on basic, translational and clinical research aimed at finding a cure for HIV and understanding the interaction between HIV and hepatitis B virus. Her laboratory is funded by the NHMRC, the National Institutes of Health, The Wellcome Trust, the American Foundation for AIDS Research and multiple commercial partnerships. She is also the Chief Investigator of a NHMRC Centre of Research Excellence (CRE), The Australian Partnership for Preparedness Research on Infectious Diseases Emergencies (APPRISE) that aims to bring together Australia’s leading experts in clinical, laboratory, and public health research to address the key components required for a rapid and effective emergency response to infectious diseases.

Panelists

Adrian Smith, PhD
Cytometry, Imaging and IT Manager
Centenary Institute

Alastair J. Sloan; BSc, PhD, PGCert, FHEA, CBiol, FRSB
FICD Head
Melbourne Dental School

Grace Chojnowski
Flow Cytometry and Imaging Facility Manager
Queensland Institute of Medical Research

CMLE Credit: 1.0

About the Presenter

Kevin Tsia, PhD
Professor
University of Hong Kong

Kevin Tsia received his PhD degree in electrical engineering at the University of California Los Angeles (UCLA) in 2009. He is currently a professor in the Department of Electrical and Electronic Engineering and the program director of the Biomedical Engineering Program at the University of Hong Kong (HKU). His research interest covers a broad range of subject matters including ultra-fast optical imaging for imaging flow cytometry and cell-based assay high-speed in-vivo brain imaging computational approaches for single-cell analysis. In 2012, he received the Early Career Award by the HK Research Grants Council in Hong Kong. He also received the Outstanding Young Research Award in 2015 at HKU as well as the 14th Chinese Science and Technology Award for Young Scientists in 2016. He is currently the RGC Research Fellow. He holds four-granted and four-pending US patents on ultrafast optical imaging technologies. He is co-founder of a start-up company commercializing the high-speed microscopy technology for cancer screening and treatment monitoring applications.

Webinar Summary

It has long been recognized that the association between the molecular genetic landscape that instructs expression of proteins and macromolecules in single cells is intrinsically linked with their biophysical properties (e.g., cell morphology, size, mass, force etc.). A growing body of evidence shows that the label-free assessment of biophysical properties of cells is an effective (or even more accurate) descriptor of cellular heterogeneity, compared to the conventional fluorescence markers, at single-cell precision. Furthermore, how molecular signatures translate into the emergent cellular biophysical properties has not been fully understood. Only with the recently advanced techniques can we now start to investigate this link.

This webinar will introduce how the synergism among single-cell imaging, microfluidics, and deep learning allows us to overcome the current limitations of single-cell biophysical phenotyping (in both instrumentation integration and new data analytic strategies). Specifically, a few high-throughput, deep-learning-powered imaging techniques will be described, as well as cytometry pipelines developed in our laboratory over the past few years. These platforms allow researchers to significantly scale the single-cell biophysical phenotyping throughput (beyond millions of cells) and enrich the phenotyping content by integrating with biochemical cell-based assay in a single-platform. Pushing the limit of biophysical phenotyping specificity and sensitivity, these techniques have been successfully employed to a number of biological research and clinical applications, including rare cancer cell detection in mouse blood, cancer cell sub-typing, targeted-drug sensitivity prediction, and so on.

Learning Objectives

• Recent advances in biophysical cytometry, especially label-free single-cell imaging, that enables studies of cellular heterogeneity at the levels of throughput, precision, specificity, and sensitivity that were once inconceivable.
  
• Advanced techniques, involving synergism among microfluidics, imaging and deep learning, that allow us to investigate deeper the link between molecular signatures and the emergent cellular biophysical properties. 

Who Should Attend

Biomedical scientists and engineers working on developments of cytometry platforms, single-cell imaging technologies, and single-cell analysis. 

CMLE Credit: 1.0

About the Presenter

Maria C. Jaimes, PhD
VP Technical Application Support
Cytek Biosciences, Inc.

Dr. Maria Jaimes earned her MD degree at the Universidad Javeriana in Colombia. Dr. Jaimes completed her postdoctoral training at Stanford University in the Department of Microbiology and Immunology. During her postdoc, she focused on characterizing the immune responses to both rotavirus and influenza viruses after natural infection and immunization. In 2005, Dr. Jaimes joined BD Biosciences, where she worked on different aspects of quality assurance and standardization of flow cytometry assays. In 2015, Dr. Jaimes joined Cytek Biosciences. She is part of the R&D team credited for developing the Aurora Full Spectrum Cytometer and has overseen the instrument characterization, verification, and development of multicolor applications. Besides her responsibilities within the R&D team, Dr. Jaimes leads the Technical Applications Support team worldwide.

Webinar Summary

This webinar will cover OMIP-069 (published in Cytometry Part A in August 2020), the first 40-color fluorescent panel using full spectrum flow cytometry to broadly phenotype much of the cellular composition of the human peripheral immune system. The panel in this OMIP has been thoroughly optimized to ensure high-quality data and well-resolved populations, enabling the description of most canonical subsets of T cells, B cells, NK cells, monocytes, and dendritic cells. Dr. Jaimes will present the journey of the technology, panel design, protocol development, data QC/QA, and data analysis which led to the successful achievement of this 40-color immune profiling panel. 

Learning Objectives

• Understand the concepts behind full spectrum profiling.
• Learn the necessary steps and tools for good panel design.
• Develop expertise on how to troubleshoot and optimize a multicolor panel.
• Learn to recognize high quality vs. compromised data and the potential sources and mitigation of errors. 

Who Should Attend

SRL staff/directors, immunologists, CRO staff, and full spectrum flow cytometer users.

CMLE Credit: 1.0

About the Presenters

Paula Niewold, PhD
Postdoctoral Researcher
Department of Infectious Diseases
Leiden University Medical Centre

Dr. Paula Niewold is an immunologist currently working as a postdoctoral researcher at the Department of Infectious Diseases at the Leiden University Medical Centre. She is interested in host-pathogen interactions and how they impact the outcome of disease. She has studied these interactions in models of cerebral malaria, West Nile virus encephalitis, psoriasis, and tuberculosis using high-dimensional flow, mass, and imaging mass cytometry. She is an ISAC Marylou Ingram Scholar.

Thomas Ashhurst, PhD
Immunologist and High-Dimensional Cytometry Specialist
Sydney Cytometry Facility
University of Sydney

Dr. Thomas Ashhurst is an immunologist and high-dimensional cytometry specialist with the Sydney Cytometry Facility at the University of Sydney. He develops and applies a range of single-cell cytometry technologies and computational analysis tools to map dynamic immune responses over time, space, and disease. In particular, he applies these approaches to the study of immunology and infectious disease, including emerging pathogens such as COVID-19, Zika virus encephalitis, and West Nile virus encephalitis. He is an ISAC Marylou Ingram Scholar.

Webinar Summary

In conventional fluorescence cytometry, each fluorophore in a panel is measured in a target detector, through the use of wide band-pass optical filters. In contrast, spectral cytometry uses a large number of detectors with narrow band-pass filters to measure a fluorophore's signal across the spectrum, creating a more detailed fluorescent signature for each fluorophore. The spectral approach shows promise in adding flexibility to panel design and improving the measurement of fluorescent signal. However, few comparisons between conventional and spectral systems have been reported to date. Here we present our findings comparing conventional and spectral approaches to cytometry—including comparisons of compensation and unmixing—and evaluate the use of spectral cytometry for immune profiling in viral diseases.

Learning Objectives

• Gain an understanding of the essential differences between conventional and spectral approaches to cytometry.
• Appreciate the differences between compensation and spectral unmixing.
• Consider applications for spectral cytometry in the context of immunological studies.

Who Should Attend

Researchers and technical staff who utilize flow, spectral, or mass cytometry in their work.

CMLE Credit: 1.0