
An overview of the course.
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).
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.
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.
⭐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.
VERY IMPORTANT; PLEASE NOTE:
The course is NOT focused on any specific software or instruments and is NOT intended to substitute hands-on training and/or certification courses. 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.
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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.