storage: object doesn't exist
Flow Cytometry - Introduction & Basics Guide

Flow Cytometry - Introduction & Basics Guide

Updated Flow Cytometry Basics Guide

Updated
Flow Cytometry Basics Guide

Practical advice to get you started in flow cytometry

Download Now

This flow cytometry guide aims to give you a basic overview of all the important aspects of flow cytometry.


With chapters on instrumentation, useful reagents, controls, experimental set up and much more, this guide enables best practice to be followed and gives practical advice on building multicolor panels with example protocols.

This guide is invaluable to beginners wanting to start flow cytometry and as a handy tool for teaching others about this powerful application.

To further assist learning we have recently added a Flow Cytometry glossary of terms to this guide.

Principles of the Flow Cytometer


A flow cytometer is composed of fluidic, optic, and electronic systems. This chapter explains the role of each system, and how they work together.

You will also learn the basics of electrostatic cell sorting.

Principles of Fluorescence


Flow cytometry is underpinned by the principles of fluorescence, which are covered in this chapter.

We also present useful fluorescent dyes for flow cytometry and explain fluorescence compensation for multicolor panels.

Data Analysis


Understanding how to analyze your data is critical for success with flow cytometry. In addition, since some analysis steps happen in real time with flow cytometry, it’s important to have a plan before you start an experiment.

In this chapter, you will learn how to place gates and regions for cell analysis, and how to backgate. We also examine the different plot types used to analyze and display flow cytometry data.

Controls in Flow Cytometry


Controls are vital in any experiment to reliably distinguish your results from background variation and nonspecific effects. Some controls are also specific to flow cytometry and should be included in every experiment.

These include unstained controls, viability controls, and compensation controls. Here, we discuss some essential controls for flow cytometry that will help you obtain publication-quality data.

Optimizing Your Experiments


One of the fundamentals of flow cytometry is the ability to measure single particles as they pass through a laser beam. However, the cytometer can measure only what is put in, and a poor sample will generally lead to poor data.

It is essential to start with the best possible sample and treat your samples as gently as possible so that you have a viable cell suspension and obtain good data.

Multicolor Panel Building Rules


Multicolor flow cytometry involves analyzing multiple fluorescent parameters in one sample. These parameters may be surface markers, intracellular markers, DNA, or a combination. In addition to using the right controls (Chapter 4), optimizing your experimental procedure (Chapter 5), and carefully preparing your samples, there are still other factors to consider to achieve meaningful results with multicolor flow cytometry.

When multiple dyes are used, signal from each dye can spill over into neighboring channels used to detect other dyes. This spillover can cause a loss in resolution and needs to be compensated for (Chapter 2). Other factors including high levels of noise caused by nonspecific staining, high background staining, and cell autofluorescence, can also contribute to reductions in sensitivity and resolution.

This chapter covers the basic rules

  • Signal resolution
  • Rules for optimized panel building
    • Experimental design
    • Instrument configuration
    • Fluorescent dye separation
    • Fluorescent dye properties
    • Antigen density
    • Marker expression patterns
    • Dump channels
    • Antibody titration
  • Panel building tools 
  • Example of conventional flow cytometry multicolor panel 
  • Full spectrum flow cytometry panel building

Common Applications and New Technology


So far in this guide, we have covered what flow cytometry involves and how to build experiments and analyze data, but what are the common uses of flow cytometry?

This chapter provides examples of flow cytometry applications, such an immunophenotyping and analysis of cellular functions like regulated cell death and cell proliferation. You will learn about novel innovations in flow cytometry, such as the integration of automation.

Common Protocols


This chapter brings you trusted protocols to help you jump straight in to your experiments.

  • Sample preparation protocol
  • Protocols for Preparing Cells for Flow Cytometry
    • Preparation of Tissue Culture Cells Stored in Liquid Nitrogen
    • Preparation of Tissue Culture Cells in Suspension
    • Preparation of Adherent Tissue Culture Cell Lines
    • Preparation of Human Peripheral Blood Mononuclear Cells
    • Preparation of Peritoneal Macrophage, Bone Marrow, Thymus, and Spleen Cells

  • Staining of Cells for Flow Cytometry 

    • Direct Immunofluorescence Staining Surface Epitopes of Cells and Blood
    • Indirect Immunofluorescence Staining of Surface Epitopes of Cells and Blood 
    • Direct Staining of Intracellular Antigens and Cytokines
    • Leucoperm Accessory Reagent Method
    • Direct Immunofluorescence Staining of Intracellular Cytokines in Blood
    • Propidium Iodide Staining of Cells for Cell Cycle Analysis
    • BrdU Staining of Cells for Cell Cycle Analysis and Apoptosis
    • Direct Immunofluorescence Staining of Cells with StarBright Dyes

Troubleshooting


If something does not work, check through the guidelines provided in Tables 9 and 10 to identify and resolve the problem.

If there are still difficulties and you have purchased any Bio-Rad reagents, our Technical Services Team (see contact details at bio-rad-antibodies.com/technical) can offer further advice.

Appendices


There can be many reasons for poor results. Here we list some of the common problems with advice to help you get the best staining possible along with recommended reading if you want more information.