Flow Cytometry Basics for the Non-Expert (eBook)

Christine Goetz, Ph.D., Jody Bonnevier, Ph.D., and Christopher Hammerbeck, Ph.D. R&D Systems, Minneapolis, Minnesota

Preface 6
Acknowledgments 9
Contents 10
About the Book 16
Chapter 1: Flow Cytometry: Definition, History, and Uses in Biological Research 17
1 Components of Flow Cytometry 17
2 Definition of Flow Cytometry 18
3 History of Flow Cytometry 20
3.1 Cellular Impedance and the Coulter Principle 20
3.2 Fluorescence-Based Flow Cytometers 21
3.3 Monoclonal and Polyclonal Antibodies 22
3.4 Fluorescent Molecules 23
3.5 Fluorescence Activated Cell Sorting (FACS) 23
4 Modern Flow Cytometers 24
4.1 Fluidic-Based Flow Cytometers 24
4.2 Acoustic-Based Flow Cytometers 25
4.3 Mass Cytometers (CyTOF) 25
References 27
Chapter 2: Physics of a Flow Cytometer 28
1 Components of Fluidic-Based Flow Cytometers 28
1.1 Fluidics 28
1.2 Optics 30
1.3 Electronics 35
2 Putting It All Together 37
3 Materials 38
3.1 Flow Antibodies 38
3.2 Cell Activation Reagents 39
3.3 Cell Purification Kits 39
3.4 Staining Buffers/Fc Block Reagents 39
References 39
Chapter 3: The Language of Flow Cytometry and Experimental Setup 40
1 Flow Data Plots and Gating 40
1.1 Flow Data Plots 40
1.2 Gating 42
2 Mean Fluorescence Intensity (MFI) and Biexponential vs. Logarithmic Scaling 42
2.1 Mean Fluorescence Intensity (MFI) 43
2.2 Logarithmic vs. Biexponential Scaling 43
3 Forward Scatter (FSC)/Side Scatter (SSC) and Doublet Exclusion 45
3.1 FSC/SSC 45
3.2 Doublet Exclusion 46
4 CS& T and Basic Experimental Setup
4.1 CS& T
4.2 Basic Experimental Setup 48
4.2.1 Experimental/Flow Panel Design 48
4.2.2 Surface and Intracellular Staining on iTregs 48
4.2.3 Experimental Setup on the Fortessa 49
4.2.4 Sample Acquisition and Analysis 49
5 Basic Concepts of Compensation 51
6 Materials 54
6.1 Flow Antibodies 54
6.2 Cell Activation Reagents 54
6.3 Cell Purification Kits 55
6.4 Staining Buffers/Fc Block Reagents 55
References 55
Chapter 4: Fluorochrome Choices for Flow Cytometry 56
1 Fluorochromes and Their Pros and Cons 56
2 Spectrum Viewers and How to Use Them 58
3 Fluorochrome Combinations to Use/Avoid Together 58
4 Laser Strength on the Fortessa vs. the Calibur 62
5 High- vs. Low-Density Cell Markers and  Fluorochrome Choice 62
6 Materials 65
6.1 Flow Antibodies 65
6.2 Staining Buffers/Fc Block Reagents 66
References 66
Chapter 5: Using the Process of Compensation to Prevent False Positive Data Caused by Fluorescence Spillover: A Practical Example 67
1 Compensation: A Practical Example 67
2 Manual Compensation by Comparing the Median Fluorescence Intensity of Cell Populations 80
3 Considerations When Performing Compensation 83
3.1 Using the Same Compensation Settings for Multiple Experiments 83
3.2 Dimming of Fluorochromes as a Result of Compensation 84
3.3 Consider the Number of Overlapping Fluorochromes 84
3.4 Consider If Compensation Is Necessary 85
3.5 Consider If Fluorochromes with High Spectral Overlap Can Be Separated on Two Different Cell Types 85
3.6 Using Cells as Single-Stain Controls vs. Beads 87
4 Materials 87
4.1 Flow Antibodies 87
4.2 Staining Buffers/Fc Block Reagents 88
References 88
Chapter 6: Primary and Secondary Antibodies and Flow Cytometry Controls 89
1 Antibody Development for Flow Cytometry 89
2 Detection of Unconjugated Primary Antibodies 92
3 Flow Cytometry Controls 97
3.1 Isotype Controls 98
3.2 Internal Lineage Controls 102
3.3 Cell Activation Controls 106
3.4 Fluorescence Minus One (FMO) 110
4 Materials 114
4.1 Flow Antibodies 114
4.2 Isotype Control Antibodies 115
4.3 Secondary Antibodies 115
4.4 Cell Activation Reagents 115
4.5 Staining Buffers/Fc Block Reagents 115
References 116
Chapter 7: Experimental Considerations with  Data Sets as Examples 117
1 Fc Receptor Blocking 117
2 Dead Cell Exclusion 122
3 Cell Number, Antibody Concentration, and Titration of Flow Antibodies 128
3.1 Cell Number 128
3.2 Antibody Concentration 130
3.3 Titrating Flow Cytometry Antibodies 131
4 Surface Staining Considerations 133
4.1 From 2 to 12 Parameter Flow Cytometry 133
4.2 Reagents to Assess Cell Proliferation 135
4.3 Tracking Cell Populations in Mice Using Congenic Markers 139
4.4 Chemokine Receptor Staining and Dependence on Temperature 140
4.5 How to Detect Degranulation in NK Cells Using a Killing Assay 142
4.6 Protein Detection Using Fluorokines 146
4.7 Dump Channels 147
5 Intracellular Staining Considerations 149
5.1 Intracellular Staining Buffers 149
5.2 Detection of Secreted Cytokines 151
5.3 Optimizing Fixation, Staining Order, and Using the Correct Intracellular Buffer System 152
5.3.1 Optimizing Fixation Percentage 153
5.3.2 Staining Order Matters When Detecting Both Surface and Intracellular Markers 154
5.3.3 Intracellular Buffer Systems for Transcription Factors and Phospho–Proteins 155
Detection of Transcription Factors: FoxP3 156
Detection of Phospho-Proteins: Phospho-Stat1 156
6 Materials 159
6.1 Flow Antibodies 159
6.2 Isotype Control Antibodies 160
6.3 Proliferation and Viability Dyes and Reagents 161
6.4 Cell Activation Reagents 161
6.5 Cell Purification Kits 161
6.6 Staining Buffers/Fc Block Reagents 161
References 162
Chapter 8: Cell Enrichment 163
1 Enrichment of Cell Populations Using Columns 163
2 Magnetic Selection Principles and Types of Selection 164
3 Fluorescence Activated Cell Sorting (FACS) 167
4 Materials 168
4.1 Flow Antibodies 168
4.2 Isotype Controls 168
4.3 Cell Purification Kits 169
4.4 Staining Buffers/Fc Block Reagents 169
Chapter 9: Surface and Intracellular Staining Protocols for Flow Cytometry 170
1 Staining Buffers for Flow Cytometry 170
1.1 Key Tips for Surface and Intracellular Staining 171
2 Surface Staining: Tips and Tricks 172
2.1 Cell Surface Staining in Whole Blood (WB) 173
2.1.1 Cell Surface Staining for Unconjugated Antibodies in Human WB 173
2.1.2 Cell Surface Staining for Directly Conjugated Antibodies in Human WB 174
2.2 Cell Surface Staining in Peripheral Blood Mononuclear Cells (PBMCs) 175
2.2.1 Cell Surface Staining for Unconjugated Antibodies in PBMCs 175
2.2.2 Cell Surface Staining for Directly Conjugated Antibodies in PBMCs 176
2.3 Cell Surface Staining for Biotinylated Antibodies 177
2.3.1 Cell Surface Staining for Biotinylated Antibodies in WB 177
2.3.2 Cell Surface Staining for Biotinylated Antibodies in PBMCs 178
2.4 Cell Surface Staining for Chemokine Receptors 179
2.5 Cell Surface Staining for LAMP Proteins 180
3 Intracellular Staining: Tips and Tricks 181
3.1 Intracellular Staining: Formaldehyde/Saponin 181
3.1.1 Formaldehyde/Saponin IC Staining in WB with Unconjugated Antibodies 181
3.1.2 Formaldehyde/Saponin IC Staining in WB with Directly Conjugated Antibodies 183
3.1.3 Formaldehyde/Saponin IC Staining in PBMCs with Unconjugated Antibodies 184
3.1.4 Formaldehyde/Saponin IC Staining in PBMCs with Directly Conjugated Antibodies 186
3.2 Intracellular Staining: FoxP3/Transcription Factor Buffer Set 187
3.2.1 FoxP3/Transcription Factor Buffer Staining in WB with Unconjugated Antibodies 187
3.2.2 FoxP3/Transcription Factor Buffer Staining in WB with Directly Conjugated Antibodies 188
3.2.3 FoxP3/Transcription Factor Buffer Staining in PBMCs with Unconjugated Antibodies 189
3.2.4 FoxP3/Transcription Factor Buffer Staining in PBMCs with Directly Conjugated Antibodies 190
3.3 Formaldehyde/Methanol Intracellular Staining 191
3.3.1 Formaldehyde/Methanol Staining for Detection of Unconjugated Phospho–Proteins in PBMCs or Stimulated Cell Populations 192
3.3.2 Formaldehyde/Methanol Staining for Detection of Directly Conjugated Phospho-Proteins in PBMCs or Stimulated Cell Populations 193
Chapter 10: Troubleshooting 195
1 What Happens If I Permeabilize My Cells Before Fixation? What Happens If I Forget to Dilute My Perm Buffer to 1×? 195
2 What Happens If I Use PE or APC-Conjugated Surface Markers Prior to Permeabilization with Methanol? 196
3 What Happens If I Stain My Cells with the Same Antibody Used for Stimulation? 197
4 How Can I Avoid Messy Staining with Costains and Secondary Antibodies of the Same Species? 199
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? 200
6 What Happens If You Use a FITC or A488 Antibody on GFP-Tagged Cells? 201
7 Should I Use FcR Block on All Cells and with All Antibodies? 202
8 Do I Need to Use PMA, Lonomycin, and Monensin (and/or Brefeldin A) for Detection of Secreted Cytokines in T Cells and Monocytes? 203
9 Can I Add My Secondary and Primary Antibody at the Same Time to Speed Up an Experiment? 204
10 Can Biotinylated Antibodies and the Streptavidin Secondary Be Added at the Same Time? 205
11 Does Volume Matter When Surface Staining Your Cells? 206
12 What Happens If I Can’t Run My Cells on the Cytometer on the Same Day that I Stain Them? 208
13 What Happens If I Accidentally Add the Wrong Antibody to My Tube? Can I Quickly Wash It Out and Reuse Those Cells? 208
14 Can Flow Cytometry Be Used to Count Cells? 210
15 Can the Method Used to Harvest Adherent Cells Affect the Ability to Detect Protein Expression by Flow Cytometry? 211
16 Materials 212
16.1 Flow Antibodies 212
16.2 Isotype Control Antibodies 213
16.3 Secondary Antibodies and Staining Reagents 214
16.4 MISC Flow Cytometry Reagents 214
16.5 Cell Culture Reagents 214
16.6 Cell Activation Reagents 214
16.7 Cell Purification Kits 214
16.8 Staining Buffers 214
References 215
Glossary 216
Index 223