Introduction To Flow Cytometry

A flow cytometry primer

What you'll learn
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
Requirements
No flow cytometry experience needed.
Description
This course is designed to help beginner or occasional users of flow cytometry become more familiar with the numerous facets of the field. 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. 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. 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.

Overview

Section 1: Theoretical foundations of flow cytometry

Lecture 1 Introduction

Lecture 2 What Is Cytometry and What Does It Do?

Lecture 3 Fluorescence Overview

Lecture 4 Excitation and Emission Spectra

Section 2: Flow Cytometers

Lecture 5 The Components of a Flow Cytometer

Lecture 6 Fluidics

Lecture 7 Optics

Lecture 8 Electronics

Lecture 9 Quality Control

Lecture 10 Maintenance

Section 3: Working with Fluorochromes

Lecture 11 Fluorochrome Overview

Lecture 12 Relative Brightness

Lecture 13 Multicolour Flow Cytometry

Lecture 14 Compensation

Lecture 15 Spreading

Section 4: Experiment Planning

Lecture 16 Experiment Preparation - General Guidelines

Lecture 17 Experimental Controls

Lecture 18 Counting and Titration

Section 5: Data Acquisition and Analysis

Lecture 19 Threshold and Doublet Discrimination

Lecture 20 Scaling, Plots and Gates

Lecture 21 Example Workflow and Good Practice Gating

Beginner or occasional users of flow cytometry.