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Introduction to Flow Cytometry

4.2 Filled star Filled star Filled star Filled star Empty star
Based on 77 ratings
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Lavinia Ionescu

⭐Average of ALL reviews received, equally weighted: 4.2 stars.⭐

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⭐Average of ALL reviews received, equally weighted: 4.2 stars.⭐

This course is designed to help beginner or occasional users of flow cytometry become more familiar with the numerous facets of the field, starting with a theoretical perspective. It may also be useful to intermediate or more frequent operators as a review of the concepts that remain firmly in place across platforms: from the basics of light scattering and fluorescence to the universal main components of flow cytometers, the practical principles of multicolor compensation, and the roles of experimental controls typically used in flow cytometry. Please feel welcome to check out the curriculum/descriptions of the individual videos for a few additional details that may offer a more accurate idea of the scope of the course.

Instead, it aims to present the elements that constitute a shared ground among platforms and provide a springboard to advanced flow cytometry concepts.

  • The course does NOT go into highly specific details regarding sample preparation, acquisition and analysis. This is due to the variety of possible applications, instruments, reagents and dedicated software, as well as the wide online availability of such protocols (often tailored to particular commercial products) for the most commonly used assays - e.g., cell phenotyping using surface vs. intracellular markers, counting, apoptosis, cell cycle, proliferation, "exotic applications" etc. The instructor is not affiliated with any commercial entity and does not endorse any particular commercially available product (instrument, reagent, software or other) in this course.

    • The instructor has 15 years of experience in flow cytometry, at first in academia as a biomedical researcher, then in the industry as a flow cytometry application scientist.

    The lectures are built with the awareness that flow cytometry is used for numerous applications, almost all of which rely upon detection of fluorescent molecules. Moreover, in today's "flow" landscape, one-laser instruments coexist with multi-laser analyzers capable of detecting dozens of colors, and several powerful options are available in terms of analysis software.

    Captions: The course has captions written from scratch (10 of 21 videos), as well as automatic captions - kindly provided by the platform - that have been reviewed and edited (where necessary) by the instructor.

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    What's inside

    Learning objectives

    • Understand the fundamental principles of flow cytometry
    • Identify the components of a flow cytometer and describe their role
    • Interpret instrument quality control data
    • Understand how to determine what fluorochromes can be detected by a flow cytometer based on fluorochrome spectra and instrument configuration
    • Summarize characteristics of fluorochromes that are highly relevant in practice (single/tandem, brightness, fluorescence spillover and spreading potential)
    • Gain a working understanding of fluorescence compensation
    • Plan a flow cytometry experiment using adequate controls
    • Learn common first steps for troubleshooting

    Syllabus

    A definition of flow cytometry, brief historical summary and fundamentals of light scattering. Students will be able to describe the significance of forward and side scatter (FS/FSC and SS/SSC).

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    An overview of the course.

    Definitions of fluorescence and related terms.

    Explanation of fluorochrome excitation and emission spectra. These concepts are highly relevant for fluorochrome (dye) choices, in conjunction with the capabilities of the flow cytometer(s) being used.

    Choose any spectral viewer available online to find the excitation and emission peaks of phycoerythrin (PE).
    A document listing several spectral viewers (all free to use) is included as a course resource (Section 1, Lecture 4).

    Note: Many instrument and reagent manufacturers offer spectral viewers. Some of the more recently developed fluorochromes may only be listed in the manufacturer's spectral viewer, but the "traditional" dyes, such as fluorescein (FITC), phycoerythrin (PE), or allophycocyanin (APC) appear in most viewers.

    Overview of the main components of a flow cytometry analyzer.

    Explanation of the role of fluidics and of the main methods to achieve single-event focus; in other words, how the sample "moves through" the instrument as information regarding individual cells is collected.

    Review of the components of the optics compartment and explanation of the term optical configuration, which is very frequently used to describe the capabilities of a flow cytometer.

    Overview of the electronic components and essential pulse characteristics.

    Review of principles underlying instrument quality control (QC). QC is run regularly to ensure that the flow cytometer performs within specifications.

    General guidelines regarding maintenance operations - keeping the flow cytometer clean is a necessary step to obtaining quality data.

    Fluorochrome sources; tandem dyes; fluorescent proteins.

    Dyes differ in terms of brightness, but the brightest dyes are not necessarily always the best choice; relevant to panel design.

    Discussion on multicolour flow cytometry, which poses unique challenges in terms of designing panels and ensuring that the collected data is accurate (by correctly assigning the collected fluorescence to the corresponding dyes - also see next lecture: Compensation).

    Review of multicolour compensation principles.

    Explanation of spreading and general guidelines to help minimize its impact when designing a multicolour panel.

    GENERAL concepts applicable to experiment preparation; no detailed, assay-specific sample preparation protocols, only aspects that may need to be considered, depending on assay of interest: permeabilization, blocking, titration (also mentioned in the last video in this section).

    Review of the types of controls used in flow cytometry.

    The basic principles of counting and titration to achieve optimal staining concentration.

    Acquiring "clean" data: role of thresholding and discriminating between singlet and doublet events.

    Elements of visual representation of the data that are common across platforms; no specific instrument/software discussed.

    Traffic lights

    Read about what's good
    what should give you pause
    and possible dealbreakers
    Provides a theoretical perspective of flow cytometry, which can help beginners build a strong foundation in the field and its applications
    Covers the basics of light scattering and fluorescence, which are essential for understanding how flow cytometers work and how to interpret data
    Explores the universal main components of flow cytometers, which helps learners understand the similarities and differences between different platforms
    Presents the elements that constitute a shared ground among platforms, which provides a springboard to advanced flow cytometry concepts
    Does not go into highly specific details regarding sample preparation, acquisition, and analysis, so learners may need to seek additional resources for specific applications
    Requires learners to choose any spectral viewer available online to find the excitation and emission peaks of phycoerythrin (PE), which may require additional effort

    Save this course

    Create your own learning path. Save this course to your list so you can find it easily later.
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    Reviews summary

    Comprehensive introduction to flow cytometry fundamentals

    According to learners, this course provides a strong foundation in the fundamental principles of flow cytometry (positive). Students particularly praise the clear explanations of instrument components (positive), the detailed coverage of fluorochromes and compensation (positive), and the insights into experiment planning and controls (positive). The instructor's extensive experience shines through (positive), making complex topics accessible. While it successfully covers general concepts applicable across various platforms (neutral), reviewers note the course explicitly does not delve into specific sample preparation protocols, acquisition details, or software usage (warning), which some learners found limiting for immediate practical application. Overall, it's considered ideal for beginners and occasional users (positive) looking to build a solid theoretical understanding.
    Insights into designing experiments.
    "The module on experiment planning and controls was very practical."
    "I learned which controls are essential for my flow cytometry assays."
    "Tips on titration and sample preparation considerations were valuable."
    Ideal starting point for new users.
    "This course is perfect for someone just starting out with flow cytometry."
    "It's exactly what I needed as an occasional user to refresh my knowledge."
    "Experienced users might find some parts too basic, but it's great for beginners."
    Components and their roles explained well.
    "The sections on instrument components like fluidics and optics were well-explained."
    "I now understand the purpose of different parts of the flow cytometer."
    "The overview of electronic components and pulse characteristics was concise yet informative."
    Complex topics made understandable.
    "The explanation of compensation was incredibly helpful; it's a topic I struggled with before."
    "Understanding fluorochrome spectra and brightness was made much clearer."
    "The discussion on spreading potential was particularly useful for panel design considerations."
    Builds strong understanding of core concepts.
    "This course provided me with a solid foundation in flow cytometry."
    "I finally understand the basic principles, which was my main goal."
    "The early lectures explaining light scattering and fluorescence were very clear and helpful."
    General theory, no detailed lab steps.
    "As the course description notes, there are no specific sample preparation protocols, which would have been useful."
    "I was hoping for more detailed guidance on data acquisition settings."
    "It's a good theoretical course, but I need to find other resources for hands-on lab techniques."

    Activities

    Be better prepared before your course. Deepen your understanding during and after it. Supplement your coursework and achieve mastery of the topics covered in Introduction to Flow Cytometry with these activities:
    Review Basic Optics Principles
    Reviewing basic optics principles will help you better understand the underlying physics of flow cytometry, especially light scattering and fluorescence.
    Show steps
    • Review your notes from previous physics or optics courses.
    • Read introductory articles or book chapters on light and optics.
    • Watch online videos explaining light scattering and fluorescence.
    Read 'Flow Cytometry for Dummies'
    Reading this book will provide a simplified overview of flow cytometry concepts.
    View Melania on Amazon
    Show steps
    • Obtain a copy of 'Flow Cytometry for Dummies'.
    • Read the chapters covering basic principles and applications.
    • Focus on understanding the key concepts and terminology.
    Read 'Practical Flow Cytometry' by Howard Shapiro
    Reading this book will provide a deeper understanding of the theoretical and practical aspects of flow cytometry.
    View Practical Flow Cytometry on Amazon
    Show steps
    • Obtain a copy of 'Practical Flow Cytometry' by Howard Shapiro.
    • Read the chapters relevant to the course topics, such as light scattering, fluorescence, and compensation.
    • Take notes on key concepts and techniques.
    Four other activities
    Expand to see all activities and additional details
    Show all seven activities
    Fluorochrome and Laser Matching Exercises
    Practicing matching fluorochromes to available lasers and filters will improve your ability to design effective flow cytometry panels.
    Show steps
    • Find online spectral viewers and instrument configuration tools.
    • Create a list of common fluorochromes and laser lines.
    • Practice matching fluorochromes to lasers based on excitation and emission spectra.
    • Check your answers against published flow cytometry panels.
    Follow Online Compensation Tutorials
    Following online tutorials will help you understand and practice fluorescence compensation techniques.
    Show steps
    • Search for online tutorials on fluorescence compensation in flow cytometry.
    • Follow the tutorials step-by-step, using your own data or sample data provided in the tutorial.
    • Practice adjusting compensation settings and observe the effects on your data.
    Create a Fluorochrome Reference Sheet
    Creating a reference sheet will help you consolidate your knowledge of fluorochromes and their properties.
    Show steps
    • Research common fluorochromes and their properties (brightness, spillover, etc.).
    • Organize the information into a table or chart.
    • Include excitation and emission spectra for each fluorochrome.
    • Share your reference sheet with other students for feedback.
    Design a Flow Cytometry Panel
    Designing a flow cytometry panel will allow you to apply your knowledge of fluorochromes, compensation, and controls.
    Show steps
    • Define a research question that can be addressed using flow cytometry.
    • Identify the cell populations and markers of interest.
    • Select fluorochromes that are compatible with your instrument and markers.
    • Design a gating strategy to identify the cell populations of interest.
    • Include appropriate controls in your panel design.

    Career center

    Learners who complete Introduction to Flow Cytometry will develop knowledge and skills that may be useful to these careers:
    Flow Cytometry Specialist
    A Flow Cytometry Specialist is deeply involved with operating and maintaining flow cytometers, often in a research or clinical setting. They use these instruments to analyze cells and particles for various applications, such as cell phenotyping and disease diagnosis. A strong grasp of the theoretical underpinnings of flow cytometry and its components is crucial for this role. This course helps build a solid foundation in these principles, covering light scattering, fluorescence, and the role of each component within the flow cytometer. Further, understanding fluorochromes, compensation, and controls will enable accurate data acquisition and experiments.
    See salaries and explore the career path for Flow Cytometry Specialist
    Research Scientist
    Research Scientists design and conduct experiments to investigate specific scientific questions, often in the fields of biology, medicine, or immunology. For those focusing on cellular analysis, flow cytometry is a powerful and indispensable technique. This course is useful because it introduces the theoretical foundations of flow cytometry, including light scattering, fluorescence, and the components of the instrument. Learning about fluorochromes, compensation, and the importance of experimental controls may assist a Research Scientist in designing robust experiments and interpreting flow cytometry data accurately.
    See salaries and explore the career path for Research Scientist
    Application Scientist
    An Application Scientist provides technical support and training to customers who use scientific instruments or reagents. Since this course instructor has practical experience as an application scientist, this course provides insight into the daily life of the job. For flow cytometry, this involves teaching customers how to operate flow cytometers, design experiments, and analyze data. A comprehensive understanding of flow cytometry principles is essential for this role. The course, which covers light scattering, fluorescence, instrument components, fluorochromes, compensation, and controls, helps build expertise in the fundamentals of flow cytometry. This may assist in effectively communicating technical information and troubleshooting issues.
    See salaries and explore the career path for Application Scientist
    Laboratory Manager
    Laboratory Managers oversee the day-to-day operations of a research or clinical laboratory, including managing equipment, personnel, and budgets. Flow cytometry is a common technique in many labs, so understanding its principles, applications, and quality control procedures is beneficial. This course helps build a strong understanding of these areas, including instrument components, optics, electronics and quality control data. Learning about maintenance operations, fluorochromes, and compensation may assist a laboratory manager in ensuring the lab's flow cytometers are well-maintained and generating reliable data.
    See salaries and explore the career path for Laboratory Manager
    Clinical Laboratory Technologist
    Clinical Laboratory Technologists perform laboratory tests to diagnose and monitor diseases. Flow cytometry is used in clinical settings for immunophenotyping, leukemia and lymphoma diagnosis, and other applications. This course helps understanding of the fundamentals of flow cytometry, including light scattering, fluorescence, and instrument components could be very useful. The course's focus on fluorochromes, compensation, and controls may assist the technologist in performing accurate and reliable flow cytometry assays.
    See salaries and explore the career path for Clinical Laboratory Technologist
    Biomedical Engineer
    Biomedical Engineers design and develop medical devices and equipment, including flow cytometers. A solid understanding of the principles behind flow cytometry is valuable for this role. This course aids in familiarizing oneself with the various facets of the field, covering light scattering, fluorescence, instrument components, and quality control. This understanding may assist in developing and improving flow cytometry technology.
    See salaries and explore the career path for Biomedical Engineer
    Cell Biologist
    Cell Biologists study the structure, function, and behavior of cells. Flow cytometry is a powerful tool for analyzing cell populations and understanding cellular processes. This course helps students become more familiar with the numerous facets of flow cytometry. The course's focus on light scattering, fluorescence, fluorochromes, and experimental controls may enable the cell biologist to design and interpret flow cytometry experiments effectively.
    See salaries and explore the career path for Cell Biologist
    Immunologist
    Immunologists study the immune system and its role in health and disease. Flow cytometry is a cornerstone technique in immunology research, used for immunophenotyping, cell sorting, and analyzing immune responses. This course explores the theoretical perspective of flow cytometry. The course's focus on fluorochromes, compensation, and experimental controls may provide the immunologist with the knowledge to design and interpret flow cytometry experiments effectively.
    See salaries and explore the career path for Immunologist
    Process Development Scientist
    Process Development Scientists focus on optimizing and scaling up biological processes for manufacturing, often in the biotechnology or pharmaceutical industries. Flow cytometry may be used to monitor cell health, productivity, and product quality during bioprocessing. This course may introduce the fundamentals of flow cytometry, including light scattering, fluorescence, and instrument components.
    See salaries and explore the career path for Process Development Scientist
    Quality Control Analyst
    Quality Control Analysts ensure that products meet specific quality standards, often in the pharmaceutical or biotechnology industries. Flow cytometry may be used to assess the quality of cell-based therapies or other biological products. This course may be useful for familiarizing oneself with instrument quality control data, fluorochromes, and compensation to ensure the accuracy of flow cytometry assays.
    See salaries and explore the career path for Quality Control Analyst
    Medical Science Liaison
    Medical Science Liaisons communicate scientific information about pharmaceutical products or medical devices to healthcare professionals. If the products they represent involve flow cytometry, understanding the technology is valuable. The course's overview of flow cytometry principles will enhance credibility when discussing the science with experts and allow for a deeper understanding of the product's applications.
    See salaries and explore the career path for Medical Science Liaison
    Research Associate
    Research Associates assist scientists in conducting experiments and analyzing data. Flow cytometry is a common technique, and familiarity with its principles is a major plus. This course helps build a foundation in the basics like light scattering, fluorescence, and instrument components. Learning about fluorochromes, compensation, and controls may assist a Research Associate in performing flow cytometry experiments, and in understanding the process of quality control.
    See salaries and explore the career path for Research Associate
    Product Manager
    Product Managers oversee the development, launch, and marketing of products, including those related to scientific instrumentation or reagents. A basic understanding of flow cytometry can be helpful if managing flow cytometry-related products. Knowing the fundamentals will facilitate market analysis, customer understanding, and communication with technical teams. Such knowledge may also assist in developing effective marketing strategies.
    See salaries and explore the career path for Product Manager
    Technical Sales Representative
    Technical Sales Representatives sell scientific instruments, reagents, or services to researchers and laboratories. For those selling flow cytometry equipment or reagents, a solid understanding of the technology is essential. This course may be useful because it helps build a general understanding of flow cytometry, including components of the instruments.
    See salaries and explore the career path for Technical Sales Representative
    Data Analyst
    Data Analysts collect, clean, and analyze data to identify trends and insights that can inform decision-making. In the context of flow cytometry, a Data Analyst may need to process and interpret large datasets generated by flow cytometry experiments. The course focuses on light scattering, fluorescence, instrument components, and quality control data and may assist the Data Analyst to develop a comprehensive understanding of flow cytometry data.
    See salaries and explore the career path for Data Analyst

    Reading list

    We've selected two books that we think will supplement your learning. Use these to develop background knowledge, enrich your coursework, and gain a deeper understanding of the topics covered in Introduction to Flow Cytometry.
    Cover image
    Practical Flow Cytometry
    Save
    Is considered the bible of flow cytometry. It provides a comprehensive overview of the principles, instrumentation, and applications of flow cytometry. It valuable resource for both beginners and experienced users, offering in-depth explanations and practical guidance. This book adds significant depth to the course material and serves as an excellent reference tool.
    Practical Flow Cytometry
    Hardcover
    $$$$
    Practical Flow Cytometry, 4th Edition (Life...
    Hardcover
    $$$$

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    Course image
    Rating
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    Effort
    2.5 total hours
    Via
    Udemy
    Instructor
    Lavinia Ionescu
    Language
    English
    Traffic lights
    Read about what's good
    what should give you pause
    and possible dealbreakers
    Provides a theoretical perspective of flow cytometry, which can help beginners build a strong foundation in the field and its applications
    Covers the basics of light scattering and fluorescence, which are essential for understanding how flow cytometers work and how to interpret data
    Explores the universal main components of flow cytometers, which helps learners understand the similarities and differences between different platforms
    Presents the elements that constitute a shared ground among platforms, which provides a springboard to advanced flow cytometry concepts
    Does not go into highly specific details regarding sample preparation, acquisition, and analysis, so learners may need to seek additional resources for specific applications
    Requires learners to choose any spectral viewer available online to find the excitation and emission peaks of phycoerythrin (PE), which may require additional effort
    Share this
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