Preface
Acknowledgments
Contents
Contributors
Chapter 1: Single Cell Isolation from Surgically Resected Tissue Via Mechanical Dissociation Using TissueGrinder
1 Introduction
2 Materials
2.1 Tissue Transport
2.2 Dicing of the Tissue
2.3 Tissue Sample Processing with the TissueGrinder
2.4 Cell Counting and Viability
2.5 Cell Seeding for Cell Culture
3 Methods
3.1 Tissue Transport
3.2 Pre-Cutting of the Tissue Sample
3.3 Processing with the TissueGrinder
3.4 Cell Counting and Viability (see Notes 9-12)
3.5 Cell Culture
4 Notes
References
Chapter 2: Circulating Tumor Cell Enrichment and Single-Cell Isolation Combining the CellSearch and DEPArray Systems
1 Introduction
2 Materials
2.1 CellSearch System (see Fig. 1), Kits, and Additional Materials Required for Cell Enrichment
2.1.1 CELLTRACKS AUTOPREP System
2.1.2 CELLTRACKS ANALYZER II
2.1.3 CellSearch Circulating Tumor Cell Kit
2.1.4 CellSearch Circulating Tumor Cell Control Kit
2.1.5 Additional Materials
2.2 Sample Preparation for (Single)-Cell Isolation with the DEPArray Platform
2.3 (Single)-Cell Isolation with the DEPArray Platform
2.4 Volume Reduction of DEPArray Isolated Cells
3 Methods
3.1 CTC Enrichment with the CELLTRACKS AUTOPREP System
3.2 CTC Counting with the CELLTRACKS ANALYZER II System
3.3 Sample Preparation for Single-Cell Isolation with the DEPArray System
3.4 Cell Isolation with the DEPArray System
3.5 Preparation of Isolated Cells for Molecular Analyses
4 Notes
References
Chapter 3: Isolation of Viable Epithelial and Mesenchymal Circulating Tumor Cells from Breast Cancer Patients
1 Introduction
2 Materials
2.1 CTCs Enrichment
2.2 Immunofluorescent Staining
2.3 CD45-Positive Cells Negative Selection
2.4 Additional Equipment
3 Methods
3.1 CTCs Enrichment
3.2 Immunofluorescent Staining and Negative Selection of CD45-Positive Cells
3.3 Single CTCs Isolation by Micromanipulation
4 Notes
References
Chapter 4: Single-Cell Recovery from Tumor Cell Xenotransplanted Zebrafish Embryos for the Study of Metastasis-Initiating Cells
1 Introduction
2 Materials
2.1 Reagents
2.1.1 Zebrafish Embryo Euthanasia and Dissociation
2.1.2 Single-Cell Micromanipulation
2.2 Equipment
2.2.1 Zebrafish Embryo Euthanasia and Dissociation
2.2.2 Single-Cell Micromanipulation
3 Methods
3.1 Zebrafish Embryo Euthanasia and Dissection (see Note 1)
3.2 Zebrafish Embryo Enzymatic and Mechanical Digestion
3.3 Preparation of Collection Chamber for a Cellular Suspension
3.4 Capillary Preparation for Micromanipulation
3.5 Cell Picking
4 Notes
References
Chapter 5: Isolation of Single Circulating Tumor Cells Using VyCAP Puncher System
1 Introduction
2 Materials
3 Methods
3.1 Recovering MAGNEST Cartridge Content
3.2 Cell Seeding
3.3 Isolation of CTCs with the Punching System
4 Notes
References
Chapter 6: Simultaneous Isolation and Amplification of mRNA and Genomic DNA of a Single Cell
1 Introduction
2 Materials
2.1 Isolation of mRNA and Genomic DNA, and Whole Transcriptome Amplification
2.1.1 Reagents and Kits
2.1.2 Plasticware
2.1.3 Equipment
2.1.4 Cycling Programs
2.1.5 Reagent Mixes
2.2 DNA Precipitation and Whole Genome Amplification
2.2.1 Reagents and Kits
2.2.2 Plasticware
2.2.3 Equipment
2.2.4 Reagent Mixes
2.2.5 Cycling Programs
2.3 Control of the WTA Quality
2.3.1 Reagents and Kits
2.3.2 Plasticware
2.3.3 Equipment
2.3.4 Reagent Mixes
2.3.5 Cycling Programs
2.4 Control of the WGA Quality
2.4.1 Reagents and Kits
2.4.2 Plasticware
2.4.3 Equipment
2.4.4 Reagent Mixes
2.4.5 Cycling Programs
3 Methods
3.1 Isolation of mRNA and Genomic DNA, and Whole Transcriptome Amplification
3.2 DNA Precipitation and Whole Genome Amplification
3.3 Checking the Quality of WTA
3.4 Checking the Quality of WGA
4 Notes
References
Chapter 7: Isolation and Genomic Analysis of Circulating Tumor Cell Clusters in Cancer Patients
1 Introduction
2 Materials
2.1 CTC-Cluster Enrichment and Collection of Single CTC Clusters (See Subheading 3.1)
2.2 Genomic Analysis of CTC Clusters: WGA, QC, and Low-Pass CNA (See Subheading 3.2)
2.2.1 Whole Genome Amplification (WGA)
2.2.2 Quality Control (QC)
2.2.3 LowPass Copy Number Alteration (LpCNA) Sequencing
2.3 Bioinformatic Analysis and Interpretation of Sequencing Data
2.3.1 Pre-processing and Alignment
2.3.2 Alignment Quality Control
2.3.3 Copy Number Alteration Analysis and Tumor Fraction Estimation
2.3.4 Data Visualization and Operative System
3 Methods
3.1 CTC-Cluster Enrichment and Picking of Single CTC Clusters
3.2 Genomic Analysis of CTC Clusters
3.2.1 Whole Genome Amplification (WGA)
3.2.2 Quality Control (QC) of WGA Products
3.2.3 Low Pass CNA Sequencing
3.3 Bioinformatic Analysis and Interpretation of Sequencing Data
3.3.1 Pre-processing and Alignment (See Note 31)
3.3.2 Alignment Quality Control
3.3.3 Copy Number Alteration Analysis and Tumor Fraction Estimation
4 Notes
References
Chapter 8: Establishing Single-Cell Clones from In Vitro-Cultured Circulating Tumor Cells
1 Introduction
2 Materials
2.1 Flow Cytometry
2.2 CTC Culture
2.3 Incubator Conditions for Single-Clone Cell Culture
3 Methods
3.1 Flow Cytometry
3.2 CTC Culture
4 Notes
References
Chapter 9: Immunofluorescence Combined with Single-Molecule RNA Fluorescence In Situ Hybridization for Concurrent Detection of...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Fixation/Post-fixation
2.3 Immunofluorescence
2.4 smRNA FISH
2.5 Imaging
3 Methods
3.1 Plating and Treatment of the Cells
3.2 Fixation
3.3 Immunofluorescence
3.4 Single-Molecule RNA Fluorescence In Situ Hybridization
3.5 Imaging
4 Notes
References
Chapter 10: Highly Multiplexed and Simultaneous Characterization of Protein and RNA in Single Cells by Flow or Mass Cytometry ...
1 Introduction
2 Materials
2.1 Labeling DPPs with Metal Isotopes for Mass Cytometry
2.2 General Materials for PLAYR
2.3 LCB for PLAYR-CyTOF (Optional)
2.4 Staining to Identify Dead Cells (Optional)
2.5 Surface Antibody Staining (Optional)
2.6 Cell Fixation and Permeabilization
2.7 PLAYR and Internal Antibody Staining
2.8 Additional Reagents Specific to Mass Cytometry
2.9 Software
3 Methods
3.1 Labeling DDPs with Metal Isotopes for Mass Cytometry
3.2 Surface Antibody Staining, Dead Cell Exclusion, and Preparing Cells for PLAYR
3.3 PLAYR and Internal Antibody Staining
3.4 Sample Preparation for Mass Cytometry
4 Notes
References
Chapter 11: Array-Based Comparative Genomic Hybridization for the Detection of Copy Number Alterations in Single Cells
1 Introduction
2 Materials
2.1 Reagents
2.2 Laboratory Setup
2.3 Safety Notes
2.4 DNA Templates
2.5 Fluorescent Labeling of Amplified DNA
2.6 Purification of the PCR/Digestion Product and Quantification
2.7 Hybridization
2.8 Washing
2.9 Scanning
3 Methods
3.1 PCR-Based Labeling Using Incorporation of Dye-Conjugated dNTPs and Tru1I Digestion
3.2 Purification of the PCR/Digestion Product and Measurement of the DNA Yield and Incorporation Rate
3.3 Hybridization
3.4 Washing
3.5 Scanning and Extraction
4 Notes
References
Chapter 12: Single Cell Micro RNA Sequencing Library Preparation
1 Introduction
2 Materials
2.1 Oligonucleotides
2.2 Reagents
2.3 Equipment
3 Methods
3.1 Lysis Buffer Preparation
3.2 Single Cell Isolation
3.3 miRNA-Seq Library Preparation
3.4 Bead Size Selection
3.5 Library Quality Control
4 Notes
References
Chapter 13: Immunoblot Analysis from Single Cells Using Milo Single-Cell Western Platform
1 Introduction
2 Materials
2.1 Materials Provided with Standard scWest Kit
2.2 Other Required Materials
3 Methods
3.1 Preparation of Buffers and Reagents
3.2 Priming and Rehydrating scWest Chips
3.3 Preparation of Single-Cell Suspension
3.4 Loading scWest Chips with Single-Cell Suspension
3.5 Running scWest Chip Using MILO Instrument
3.6 Probing scWest Chips with Primary and Fluorescent Secondary Antibodies
3.7 Imaging of scWest Chips
3.8 Data Analysis Using Scout Software
3.9 Limitations
4 Notes
References
Chapter 14: Imaging of Subcellular Distribution of Platinum in Single Cells Using Laser Ablation Inductively Coupled Plasma Ma...
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.3 Other Consumables
3 Methods
3.1 Preparation of Cells for Analysis
3.2 Setting Up the LA-ICP-MS System
3.3 LA-ICP-MS Analysis of Pt Content in Whole Cells
3.4 LA-ICP-MS Imaging of Pt Distribution Across Cells
3.5 Generation of LA-ICP-MS Images
4 Notes
References
Chapter 15: Patch-seq: Multimodal Profiling of Single-Cell Morphology, Electrophysiology, and Gene Expression
1 Introduction
2 Materials
2.1 Electrophysiology and Single-Cell RNA Sample Collection
2.2 cDNA Library Preparation and Sequencing
2.3 Immunohistochemistry and Morphological Recovery
3 Methods
3.1 Electrophysiology and Single-Cell RNA Sample Collection
3.2 cDNA Library Preparation and Sequencing
3.3 Immunohistochemistry and Morphological Recovery
4 Notes
References
Chapter 16: Single-Nucleus ATAC-seq for Mapping Chromatin Accessibility in Individual Cells of Murine Hearts
1 Introduction
2 Materials
3 Methods
3.1 A Brief Protocol for Cardiac Nuclei Isolation
3.2 Brief Description of the Chromium Next GEM Single-Cell ATAC Protocol
3.3 NGS Library Quality Assessment
3.4 Sequencing of the snATAC-seq Libraries
3.5 Description of the Conventional Bioinformatical Analysis Steps
4 Notes
References
Index