Preface
Contents
Contributors
Chapter 1: Immunochemistry Analysis Using Chromogenic Substrates on Tissue Sections
1 Introduction
2 Materials
2.1 Tissue Fixation
2.2 Tissue Embedding, Sectioning, Deparaffinization, Rehydration, and/or Dehydration
2.3 Antigen Retrieval Solutions (see Note 5)
2.3.1 Heat-Induced Antigen Retrieval Solutions (HIER)
2.3.2 Protease-Induced Antigen Retrieval (PIER)
2.4 Antigen Retrieval Equipment
2.5 Tissue permeabilization and Blocking
2.5.1 Hydrophobic Pen: (e.g., Dako Pen)
2.5.2 Permeabilization Buffer (for Detection of Intracellular Antigens)
2.5.3 Endogenous Activity blocking (see Note 6)
2.6 Chromogens and Substrates
3 Methods
3.1 Tissue Fixation and Processing
3.2 Antigen Retrieval
3.2.1 Heat-Induced Epitope Retrieval
3.2.2 Protease-Induced Epitope Retrieval
3.3 Permeabilization
3.4 Blocking
3.5 Immunohistochemical Staining
4 Notes
References
Chapter 2: Indirect Immunofluorescence of Tissue Sections
1 Introduction
2 Materials
2.1 Tissue Fixation
2.2 Tissue De-paraffinization
2.3 Antigen Retrieval
2.4 Tissue Permeabilization
2.5 Tissue Blocking and Antibody Incubations
2.6 Slide Mounting
3 Methods
3.1 Tissue Fixation
3.2 Tissue De-paraffinization
3.3 Antigen Retrieval
3.4 Permeabilization
3.5 Tissue Blocking and Antibody Incubations
3.6 Slide Mounting
4 Notes
References
Chapter 3: Flow Cytometry for Beginners: Hints and Tips for Approaching the Very First Single-Cell Technique
1 Introduction
2 Materials
2.1 Cell Preparation
2.2 Selection of the Best Panel
2.3 Staining of Cells
2.4 Facs Analyzer Setup
2.5 Data Acquisition and Data Analysis
3 Methods
3.1 Cell Preparation
3.1.1 For Adherent Cells
3.1.2 For Cells in Suspension
3.1.3 For Tissues
3.2 Selection of the Best Antibody Panel
3.3 Controls Preparation
3.4 Staining of Cells
3.4.1 For Surface Protein Staining (Where Permeabilization Is Not Required)
3.4.2 For Intracellular Staining
3.5 Instrument Setup and Data Acquisition
3.6 Data Analysis
4 Notes
References
Chapter 4: High-Dimensional Immunophenotyping with 37-Color Panel Using Full-Spectrum Cytometry
1 Introduction
2 Materials
2.1 Buffers and Reagents
2.2 Monoclonal Antibodies
2.3 Consumables and Equipment
3 Methods
3.1 Lysis of Red Blood Cells (see Note 1)
3.2 Cell Staining
3.2.1 Dead Cells Staining
3.2.2 Immunostaining
3.3 Spectral Cytometer Setup and Data Analysis
4 Notes
References
Chapter 5: Detection of Cytokine-Secreting Cells by Enzyme-Linked Immunospot (ELISpot)
1 Introduction
2 Materials
2.1 Plate Coating
2.2 Cell Culture
2.3 Detection and Analysis of Spots
3 Methods
3.1 Pre-wetting of the Membrane and Addition of Capture Antibody
3.2 Blocking/Conditioning of the Plate Wells
3.3 Stimuli
3.4 Addition of Cells
3.5 Detection of Spots (Footprint of Analyte-Secreting Cells)
3.5.1 Primary Reagent
3.5.2 Secondary Reagent
3.6 Analysis of the Plate
4 Notes
References
Chapter 6: Detection and Enumeration of Cytokine-Secreting Cells by FluoroSpot
1 Introduction
2 Materials
2.1 Plate Coating
2.2 Cell Culture
2.3 Detection and Analysis of Spots
3 Methods
3.1 Pre-wetting of the Membrane and Addition of Capture Antibody
3.2 Blocking/Conditioning of the Plate Wells
3.3 Stimuli
3.4 Addition of Cells
3.5 Detection of Spots (Footprint of Analyte-Secreting Cells)
3.5.1 Primary Reagents
3.5.2 Secondary Reagents
3.6 Analysis of the Plate
4 Notes
References
Chapter 7: Single-Cell Protein Profiling by Microdroplet Barcoding and Next-Generation Sequencing
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Antibody-DNA Conjugation
2.3 Cell staining and in-Droplet Lysis
2.4 Preparation of Barcode Droplets
2.5 Droplet Merge and SOE-PCR
2.6 Library Preparation and NGS
3 Methods
3.1 Antibody-DNA Conjugation
3.2 Cell Staining and In-Droplet Cell Lysis
3.3 Preparation of Barcode Droplets
3.4 Droplet Merge and SOE-PCR
3.5 Sequencing and Data Analysis
4 Notes
References
Chapter 8: Single Cell Proteomics Using Multiplexed Isobaric Labeling for Mass Spectrometric Analysis
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers and Solutions
2.3 Equipment and Consumables
2.4 Software for Data Analysis
3 Methods
3.1 Cell Culture
3.2 Plates Preparation and Flow Cytometry for SCP Analysis
3.3 Cell Lysate, Protein Extraction, Denaturation, and Digestion
3.4 TMTpro Labeling
3.5 Pooling of TMT Labeled Peptides
3.6 LC-MS/MS Analysis
3.7 Bulk Proteomics Analysis
3.8 Data Analysis
3.8.1 Protein Search and Identifications
3.8.2 Statistical Analysis of Bulk Proteome Data
3.8.3 Statistical Analysis of SCP Data
4 Notes
References
Chapter 9: Customizing Maxpar Direct Immune Profiling Assay with Additional Surface Marker and Intracellular Cytokine Staining...
1 Introduction
2 Materials
2.1 PBMC Isolation and Tissue Culture Reagents
2.2 Cell Staining and Acquisition Reagents
2.3 Preparation Before Starting
3 Methods
3.1 PBMC Sample Preparation
3.1.1 PBMC Isolation and Cryopreservation
3.1.2 PBMC Thawing and Viability
3.1.3 Multi-protocol Experiment Stimulation
3.1.4 Biological Replicate Study Stimulation
3.2 Cell Staining
3.2.1 Prepare PBMC
3.2.2 FcR-Block Cells
3.2.3 The Maxpar Direct Immune Profiling Assay and Surface Antibody Staining
3.2.4 Fix Cells
3.2.5 Intracellular Cytokine Staining (Optional-Skip to 3.2.7 Stain Cells with Cell-ID Intercalator-Ir, Step 1 to Forego Cytop...
3.2.6 Fresh Fix Stained and Permeabilized Cells
3.2.7 Stain Cells with Cell-ID Intercalator-Ir
3.3 Acquire Samples on Helios Mass Cytometer
3.3.1 Final Sample Processing
3.3.2 Sample Acquisition
3.3.3 Data Processing
4 Notes
References
Chapter 10: High-Dimensional Tissue Profiling by Multiplexed Ion Beam Imaging
1 Introduction
2 Materials
2.1 Deparaffinization and Rehydration
2.2 Heat-Induced Epitope Retrieval (HIER)
2.3 Staining
3 Methods
3.1 Antibody Validation and Calibration
3.2 Tissue Sections
3.3 Staining
4 Notes
References
Chapter 11: Ultra-Sensitive Quantification of Protein and mRNA in Single Mammalian Cells with Digital PLA
1 Introduction
2 Materials
2.1 Antibody Storage and PLA Probe Conjugation
2.2 PLA
2.3 Droplet Digital PCR
2.4 Microfluidic Chip Fabrication
2.5 ΞΌ-dPLA
3 Methods
3.1 Antibody Storage
3.2 PLA Probe Conjugation
3.3 PLA Probe-Antigen Incubation
3.4 PLA Ligation
3.5 Droplet Digital PCR
3.6 PLA Probe Conjugation Validation
3.7 Microfluidic Device Fabrication
3.8 ΞΌ-dPLA Procedure
4 Notes
References
Chapter 12: High-Throughput Multimodal Single-Cell Targeted DNA and Surface Protein Analysis Using the Mission Bio Tapestri Pl...
1 Introduction
2 Materials
2.1 Single-Cell Isolation and DNA Library Preparation with Tapestri
2.2 Cell Staining and Protein Library Preparation
2.3 Reagent Mixes
2.4 Consumables and Equipment
3 Methods
3.1 Cell Preparation
3.2 Cell Encapsulation and Lysis
3.3 Cell Barcoding and Targeted PCR
3.4 DNA Library Preparation
3.5 Protein Library Preparation
4 Notes
References
Chapter 13: Simultaneous Analysis of Single-Cell Transcriptomes and Cell Surface Protein Expression of Human Hematopoietic Ste...
1 Introduction
2 Materials
2.1 Thawing Cells
2.2 CD34 Enrichment
2.3 Staining with TotalSeq and Fluorochrome-Conjugated Antibodies
2.4 Droplet-Based scRNA-Seq Using the 10x Genomics Platform
3 Methods
3.1 Thawing Samples
3.2 CD34 Enrichment Using CD34 MicroBeads (see Note 13)
3.3 Cell Surface Staining with TotalSeq and Fluorochrome-Conjugated Antibodies, Fluorescent-Activated Cell Sorting (FACS)
3.4 Droplet-Based scRNA-Seq Using the 10x Genomics Platform
4 Notes
References
Chapter 14: Generation of Centered Log-Ratio Normalized Antibody-Derived Tag Counts from Large Single-Cell Sequencing Datasets
1 Introduction
2 Materials
3 Methods
3.1 Preparing the AWS System with RStudio Server
3.2 Preparing the Sample Dataset
3.3 Demultiplexing Data into Singlets
3.4 Visualizing CLR Normalized and Log Normalized ADT Data
4 Notes
References
Chapter 15: Simultaneous Quantification of Single-Cell Proteomes and Transcriptomes in Integrated Fluidic Circuits
1 Introduction
2 Materials
2.1 Reagent Preparation
2.2 Materials to Label Cells with Ab BC and Run on C1
2.3 Materials to Run Single-cell Lysis, Reverse Transcription, and Preamp on C1
2.4 Materials to Prepare Ab BC Amplicons for Sequencing
2.5 Materials to Prepare cDNA Amplicons for Sequencing
3 Methods
3.1 Cell Preparation for Cell Loading Onto C1
3.2 IFC Priming on the C1
3.3 IFC Cell Loading on the C1
3.4 Cell Imaging for Quality Control
3.5 Single-Cell Lysis, Reverse Transcription, and Preamplification on C1
3.6 Amplicon Harvest and Cleanup
3.7 Amplicon Separation of cDNA and Ab BC
3.8 First Cleanup of cDNA
3.9 Ab BC Amplicon Preparation for Sequencing
3.10 Removal of Unbound Barcodes from Ab BC
3.11 Addition of Custom Primers to Ab BC
3.12 Post-indexing Cleanup of Ab BC
3.13 Ab BC Libraries Quantification and Dilution
3.14 Preparation of cDNA Amplicons for Sequencing
3.15 cDNA Library Quantification and Dilution
3.16 cDNA Tagmentation
3.17 cDNA Library Pooling and Cleanup
3.18 Pooled cDNA Library Size Distribution and Quantification
3.19 Next-Generation Sequencing Parameters
3.20 Next-Generation Sequencing Data Analysis
4 Notes
References
Chapter 16: Combined Measurement of RNA and Protein Expression on a Single-Cell Level
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 BD Rhapsody Cartridge Priming
2.3 Sample Tags and BD AbSeq AbOs Labeling
2.4 Cell Counting and Viability Measurement
2.5 BD Single-Cell Capture and cDNA Synthesis
2.6 BD mRNA Whole Transcriptome Analysis (WTA)
2.7 Master Mixes
3 Methods [11-13]
3.1 BD Rhapsody Cartridge Priming
3.2 Sample Tags and BD AbSeq AbOs Labeling
3.3 Staining with Viability Markers, Counting and Preparation of Single-Cell Suspension for Cartridge Loading
3.4 Cell Loading in the Cartridge
3.5 Preparation and Loading of Cell Capture Beads
3.6 Lysing Cells
3.7 Retrieving and Washing of Cell Capture Beads
3.8 Performing Reverse Transcription
3.9 Exonuclease I Treatment
3.10 Random Priming and Extension (RPE) on BD Rhapsody Cell Capture Beads with cDNA (see Note 15)
3.11 RPE Product Purification
3.12 Performing RPE PCR
3.13 RPE PCR Amplification Product Purification
3.14 AbSeq/Sample Tag PCR1
3.15 AbSeq/Sample Tag PCR1 Products Purification
3.16 Performing Sample Tag PCR2 on the AbSeq/Sample Tag PCR1 Product
3.17 Sample Tag PCR2 Product Purification
3.18 Performing Sample Tag Index PCR
3.19 Sample Tag Index PCR Products Purification
3.20 Performing WTA Index PCR
3.21 Dual-Sided Cleanup WTA Index PCR Product Purification
3.22 Additional WTA Index PCR Purification Steps
3.23 Performing AbSeq Index PCR
3.24 AbSeq Index PCR Products Purification
4 Notes
References
Chapter 17: Simultaneous DNA, RNA, and Protein Analysis from Single Cells Using a High-Throughput Microfluidic Workflow for Re...
1 Introduction
2 Materials
2.1 Single-Cell Isolation and DNA Library Preparation with Tapestri
2.2 Cell Staining and Protein Library Preparation
2.3 RNA Library Preparation
2.4 Reagent Mixes
2.5 Consumables and Equipment
3 Methods
3.1 Cell Staining
3.2 Encapsulation
3.3 Barcoding
3.4 Library Preparation
4 Notes
References
Correction to: High-Dimensional Immunophenotyping with 37-Color Panel Using Full-Spectrum Cytometry
Index