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Flow Cytometry Basics for the Non-Expert

Part of the Techniques in Life Science and Biomedicine for the Non-Expert Book Series

DOI: 10.1007/978-3-319-98071-3

ISBN: 978-3-319-98070-6

Publisher: Springer International Publishing - Springer Journals

Nov 9, 2018

1 - 10 of 10 Chapters

LitStream

Chapter 1Flow Cytometry: Definition, History, and Uses in Biological Research

Bonnevier, Jody; Hammerbeck, Christopher; Goetz, Christine

Pages: 1 –11

[Flow cytometry, or the analysis of cells in a fluid phase, is a powerful single-cell analysis technique used in nearly every aspect of cell biology research and clinical patient cell analysis. The development of flow cytometry began with the desire to analyze and categorize cells according to...
LitStream

Chapter 2Physics of a Flow Cytometer

Bonnevier, Jody; Huh, Jae-Bong; Hammerbeck, Christopher; Goetz, Christine

Pages: 13 –24

[Now that you are familiar with the various types of flow cytometers, we will go over how the most common type of flow cytometer, fluidic-based flow cytometers, works. In this chapter, we will touch on the physics of flow cytometry and how photons of light from fluorochromes are translated into...
LitStream

Chapter 3The Language of Flow Cytometry and Experimental Setup

Goetz, Christine; Hammerbeck, Christopher

Pages: 25 –40

[The language of flow cytometry inadvertently gives the impression that this technique is difficult. However, it is not; there is just a lot of jargon to learn like in any other field. In this chapter, we will define basic terminology that will enable end users to understand concepts important...
LitStream

Chapter 4Fluorochrome Choices for Flow Cytometry

Goetz, Christine; Hammerbeck, Christopher

Pages: 41 –51

[Multicolor flow cytometry requires thorough knowledge not only of your proteins of interest, but also of the corresponding fluorochromes that will be used in your panel. Not all fluorochromes are bright, some are prone to photobleaching, and others are sensitive to fixation and permeabilization....
LitStream

Chapter 5Using the Process of Compensation to Prevent False Positive Data Caused by Fluorescence Spillover: A Practical Example

Hammerbeck, Christopher; Goetz, Christine; Peng, Li Jen

Pages: 53 –74

[When fluorochromes are excited by a laser, they emit photons of light at a range of wavelengths in what is known as that fluorochromes emission spectrum. A portion of this emission spectrum is detected or is “seen” by the flow cytometer. However, when the emission spectrums of multiple...
LitStream

Chapter 6Primary and Secondary Antibodies and Flow Cytometry Controls

Hammerbeck, Christopher; Goetz, Christine; Bonnevier, Jody

Pages: 75 –102

[Flow cytometry requires robust, sensitive, and specific monoclonal antibodies conjugated to bright, stable fluorochromes. In this chapter, we’ll discuss the development process to generate antibodies specifically geared for use in flow cytometry. We will delve into how this technique is used for...
LitStream

Chapter 7Experimental Considerations with Data Sets as Examples

Hammerbeck, Christopher; Goetz, Christine; Peng, Li Jen; Huh, Jae-Bong

Pages: 103 –148

[Now that you have a basic understanding of the principles of flow cytometry, we will delve into how powerful this technique is for detecting multiparameter surface marker expression on cells. Accurate and clean looking data is always the goal, and we’ll show how to achieve this using dead cell...
LitStream

Chapter 8Cell Enrichment

Goetz, Christine; Hammerbeck, Christopher; Wyman, Andrew; Huh, Jae-Bong

Pages: 149 –155

[Cell enrichment is the process of purifying cells for downstream applications such as qRT-PCR, cell polarizations ex vivo, or to enrich cells for use in a flow cytometry experiment. This can be accomplished using antibody cocktails or by physically sorting out the cell populations of interest...
LitStream

Chapter 9Surface and Intracellular Staining Protocols for Flow Cytometry

Goetz, Christine; Hammerbeck, Christopher

Pages: 157 –181

[The goal of flow cytometry is to quantitate protein expression on cells or classify cells based on the expression of a particular protein. This technique is widespread as it is used in both basic developmental biology labs and the clinic. In either case, the cells need to be processed into a...
LitStream

Chapter 10Troubleshooting

Hammerbeck, Christopher; Goetz, Christine

Pages: 183 –203

[Throughout this book, we have covered many topics associated with flow cytometry. We have outlined the theory behind flow cytometry and taken you from experimental setup to analyzing your data. We’ve focused on fluorochrome choice, and how to interpret data plots post-acquisition. The...

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