Flow Cytometry Basics for the Non-Expert

Preface

Acknowledgments

Contents

About the Book

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

1 Components of Flow Cytometry

2 Definition of Flow Cytometry

3 History of Flow Cytometry

3.1 Cellular Impedance and the Coulter Principle

3.2 Fluorescence-Based Flow Cytometers

3.3 Monoclonal and Polyclonal Antibodies

3.4 Fluorescent Molecules

3.5 Fluorescence Activated Cell Sorting (FACS)

4 Modern Flow Cytometers

4.1 Fluidic-Based Flow Cytometers

4.2 Acoustic-Based Flow Cytometers

4.3 Mass Cytometers (CyTOF)

References

Chapter 2: Physics of a Flow Cytometer

1 Components of Fluidic-Based Flow Cytometers

1.1 Fluidics

1.2 Optics

1.3 Electronics

2 Putting It All Together

3 Materials

3.1 Flow Antibodies

3.2 Cell Activation Reagents

3.3 Cell Purification Kits

3.4 Staining Buffers/Fc Block Reagents

References

Chapter 3: The Language of Flow Cytometry and Experimental Setup

1 Flow Data Plots and Gating

1.1 Flow Data Plots

1.2 Gating

2 Mean Fluorescence Intensity (MFI) and Biexponential vs. Logarithmic Scaling

2.1 Mean Fluorescence Intensity (MFI)

2.2 Logarithmic vs. Biexponential Scaling

3 Forward Scatter (FSC)/Side Scatter (SSC) and Doublet Exclusion

3.1 FSC/SSC

3.2 Doublet Exclusion

4 CS&T and Basic Experimental Setup

4.1 CS&T

4.2 Basic Experimental Setup

4.2.1 Experimental/Flow Panel Design

4.2.2 Surface and Intracellular Staining on iTregs

4.2.3 Experimental Setup on the Fortessa

4.2.4 Sample Acquisition and Analysis

5 Basic Concepts of Compensation

6 Materials

6.1 Flow Antibodies

6.2 Cell Activation Reagents

6.3 Cell Purification Kits

6.4 Staining Buffers/Fc Block Reagents

References

Chapter 4: Fluorochrome Choices for Flow Cytometry

1 Fluorochromes and Their Pros and Cons

2 Spectrum Viewers and How to Use Them

3 Fluorochrome Combinations to Use/Avoid Together

4 Laser Strength on the Fortessa vs. the Calibur

5 High- vs. Low-Density Cell Markers and  Fluorochrome Choice

6 Materials

6.1 Flow Antibodies

6.2 Staining Buffers/Fc Block Reagents

References

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

1 Compensation: A Practical Example

2 Manual Compensation by Comparing the Median Fluorescence Intensity of Cell Populations

3 Considerations When Performing Compensation

3.1 Using the Same Compensation Settings for Multiple Experiments

3.2 Dimming of Fluorochromes as a Result of Compensation

3.3 Consider the Number of Overlapping Fluorochromes

3.4 Consider If Compensation Is Necessary

3.5 Consider If Fluorochromes with High Spectral Overlap Can Be Separated on Two Different Cell Types

3.6 Using Cells as Single-Stain Controls vs. Beads

4 Materials

4.1 Flow Antibodies

4.2 Staining Buffers/Fc Block Reagents

References

Chapter 6: Primary and Secondary Antibodies and Flow Cytometry Controls

1 Antibody Development for Flow Cytometry

2 Detection of Unconjugated Primary Antibodies

3 Flow Cytometry Controls

3.1 Isotype Controls

3.2 Internal Lineage Controls

3.3 Cell Activation Controls

3.4 Fluorescence Minus One (FMO)

4 Materials

4.1 Flow Antibodies

4.2 Isotype Control Antibodies

4.3 Secondary Antibodies

4.4 Cell Activation Reagents

4.5 Staining Buffers/Fc Block Reagents

References

Chapter 7: Experimental Considerations with  Data Sets as Examples

1 Fc Receptor Blocking

2 Dead Cell Exclusion

3 Cell Number, Antibody Concentration, and Titration of Flow Antibodies

3.1 Cell Number

3.2 Antibody Concentration

3.3 Titrating Flow Cytometry Antibodies

4 Surface Staining Considerations

4.1 From 2 to 12 Parameter Flow Cytometry

4.2 Reagents to Assess Cell Proliferation

4.3 Tracking Cell Populations in Mice Using Congenic Markers

4.4 Chemokine Receptor Staining and Dependence on Temperature

4.5 How to Detect Degranulation in NK Cells Using a Killing Assay

4.6 Protein Detection Using Fluorokines

4.7 Dump Channels

5 Intracellular Staining Considerations

5.1 Intracellular Staining Buffers

5.2 Detection of Secreted Cytokines

5.3 Optimizing Fixation, Staining Order, and Using the Correct Intracellular Buffer System

5.3.1 Optimizing Fixation Percentage

5.3.2 Staining Order Matters When Detecting Both Surface and Intracellular Markers

5.3.3 Intracellular Buffer Systems for Transcription Factors and Phospho–Proteins

Detection of Transcription Factors: FoxP3

Detection of Phospho-Proteins: Phospho-Stat1

6 Materials

6.1 Flow Antibodies

6.2 Isotype Control Antibodies

6.3 Proliferation and Viability Dyes and Reagents

6.4 Cell Activation Reagents

6.5 Cell Purification Kits

6.6 Staining Buffers/Fc Block Reagents

References

Chapter 8: Cell Enrichment

1 Enrichment of Cell Populations Using Columns

2 Magnetic Selection Principles and Types of Selection

3 Fluorescence Activated Cell Sorting (FACS)

4 Materials

4.1 Flow Antibodies

4.2 Isotype Controls

4.3 Cell Purification Kits

4.4 Staining Buffers/Fc Block Reagents

Chapter 9: Surface and Intracellular Staining Protocols for Flow Cytometry

1 Staining Buffers for Flow Cytometry

1.1 Key Tips for Surface and Intracellular Staining

2 Surface Staining: Tips and Tricks

2.1 Cell Surface Staining in Whole Blood (WB)

2.1.1 Cell Surface Staining for Unconjugated Antibodies in Human WB

2.1.2 Cell Surface Staining for Directly Conjugated Antibodies in Human WB

2.2 Cell Surface Staining in Peripheral Blood Mononuclear Cells (PBMCs)

2.2.1 Cell Surface Staining for Unconjugated Antibodies in PBMCs

2.2.2 Cell Surface Staining for Directly Conjugated Antibodies in PBMCs

2.3 Cell Surface Staining for Biotinylated Antibodies

2.3.1 Cell Surface Staining for Biotinylated Antibodies in WB

2.3.2 Cell Surface Staining for Biotinylated Antibodies in PBMCs

2.4 Cell Surface Staining for Chemokine Receptors

2.5 Cell Surface Staining for LAMP Proteins

3 Intracellular Staining: Tips and Tricks

3.1 Intracellular Staining: Formaldehyde/Saponin

3.1.1 Formaldehyde/Saponin IC Staining in WB with Unconjugated Antibodies

3.1.2 Formaldehyde/Saponin IC Staining in WB with Directly Conjugated Antibodies

3.1.3 Formaldehyde/Saponin IC Staining in PBMCs with Unconjugated Antibodies

3.1.4 Formaldehyde/Saponin IC Staining in PBMCs with Directly Conjugated Antibodies

3.2 Intracellular Staining: FoxP3/Transcription Factor Buffer Set

3.2.1 FoxP3/Transcription Factor Buffer Staining in WB with Unconjugated Antibodies

3.2.2 FoxP3/Transcription Factor Buffer Staining in WB with Directly Conjugated Antibodies

3.2.3 FoxP3/Transcription Factor Buffer Staining in PBMCs with Unconjugated Antibodies

3.2.4 FoxP3/Transcription Factor Buffer Staining in PBMCs with Directly Conjugated Antibodies

3.3 Formaldehyde/Methanol Intracellular Staining

3.3.1 Formaldehyde/Methanol Staining for Detection of Unconjugated Phospho–Proteins in PBMCs or Stimulated Cell Populations

3.3.2 Formaldehyde/Methanol Staining for Detection of Directly Conjugated Phospho-Proteins in PBMCs or Stimulated Cell Populations

Chapter 10: Troubleshooting

1 What Happens If I Permeabilize My Cells Before Fixation? What Happens If I Forget to Dilute My Perm Buffer to 1×?

2 What Happens If I Use PE or APC-Conjugated Surface Markers Prior to Permeabilization with Methanol?

3 What Happens If I Stain My Cells with the Same Antibody Used for Stimulation?

4 How Can I Avoid Messy Staining with Costains and Secondary Antibodies of the Same Species?

5 Why I Am Not Seeing NK1.1 Expression on Balb/c Mouse Splenocytes? Is CD14 Universally Expressed in Human and Mouse Monocytes? How About Other T and B Lineage Subsets?

6 What Happens If You Use a FITC or A488 Antibody on GFP-Tagged Cells?

7 Should I Use FcR Block on All Cells and with All Antibodies?

8 Do I Need to Use PMA, Lonomycin, and Monensin (and/or Brefeldin A) for Detection of Secreted Cytokines in T Cells and Monocytes?

9 Can I Add My Secondary and Primary Antibody at the Same Time to Speed Up an Experiment?

10 Can Biotinylated Antibodies and the Streptavidin Secondary Be Added at the Same Time?

11 Does Volume Matter When Surface Staining Your Cells?

12 What Happens If I Can’t Run My Cells on the Cytometer on the Same Day that I Stain Them?

13 What Happens If I Accidentally Add the Wrong Antibody to My Tube? Can I Quickly Wash It Out and Reuse Those Cells?

14 Can Flow Cytometry Be Used to Count Cells?

15 Can the Method Used to Harvest Adherent Cells Affect the Ability to Detect Protein Expression by Flow Cytometry?

16 Materials

16.1 Flow Antibodies

16.2 Isotype Control Antibodies

16.3 Secondary Antibodies and Staining Reagents

16.4 MISC Flow Cytometry Reagents

16.5 Cell Culture Reagents

16.6 Cell Activation Reagents

16.7 Cell Purification Kits

16.8 Staining Buffers

References

Glossary

Index