Preface
Contents
Contributors
Chapter 1: Vaccine Design by Reverse Vaccinology and Machine Learning
1 Introduction
2 Vaxign-ML
2.1 Vaxign-ML Stand-Alone Web Tool
2.2 Vaxign-ML Stand-Alone Command-Line Tool
2.3 SARS-CoV-2 Example
3 Vaxign with Vaxign-ML Integration
3.1 Vaxign Main Interface
3.2 Vaxign Analyses After Vaccine Candidate Prediction
3.3 Vaxign Pangenome Analysis
3.4 Prediction of Brucella Vaccine Antigens Using Vaxign and Vaxign-ML
4 Notes
References
Chapter 2: Application of Reverse Vaccinology and Immunoinformatic Strategies for the Identification of Vaccine Candidates Aga...
1 Introduction
2 Materials
3 Methods
3.1 Identification of Vaccine Targets Using Vaxign
3.1.1 Select a Genome
3.1.2 Select Filter Options
3.2 Identification of Conserved Identity with Other Shigella Strains
3.3 Identification of Human Homologs
3.4 Antigenicity Analysis
3.5 Prediction of Linear B-Cell Epitopes
3.6 Prediction of HLA Class I and Class II T-Cell Epitope
4 Notes
References
Chapter 3: Purification of Prospective Vaccine Antigens from Gram-Positive Pathogens by Immunoprecipitation
1 Introduction
2 Materials
2.1 Preparation of Gram-Positive Cell Wall Extracts
2.2 Coupling of Proteins to Cyanogen Bromide Activated Sepharose
2.3 Affinity Purification of Pathogen-Reactive Antibodies and Immunoprecipitation of Prospective Vaccine Antigens
3 Methods
3.1 Preparation of Gram-Positive Cell Wall Extracts
3.2 Coupling the Cell Wall Proteins to CBr-Sepharose
3.3 Affinity Purification of Pathogen-Reactive Antibodies
3.4 Coupling of Pathogen-Reactive Antibodies to CBr-Sepharose and Immunoprecipitation of Prospective Vaccine Antigens
4 Notes
References
Chapter 4: Rapid Surface Shaving for Proteomic Identification of Novel Surface Antigens for Vaccine Development
1 Introduction
2 Materials
2.1 Preparation of B. pertussis Cells
2.2 Rapid Surface Shaving
2.3 Flow Cytometry
2.4 C18 Clean Up and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
2.5 Bioinformatic Analysis and Identification of Surface Proteins
3 Methods
3.1 Preparation of B. pertussis Cells
3.2 Rapid Surface Shaving with Trypsin
3.3 Determining Cell Viability After Rapid Surface Shaving Using Propidium Iodide (PI).
3.4 C18 Peptide Clean-Up and LC-MS/MS
3.5 Bioinformatics Analysis and Identification of Surface Proteins
4 Notes
References
Chapter 5: Two-Dimensional Electrophoresis Coupled with Western Blot as a Method to Detect Potential Neutralizing Antibody Tar...
1 Introduction
2 Materials
2.1 Selection of L. intracellularis Proteins that Interact with Intestinal Pig Epithelial Cells (IPEC)
2.2 Rehydration of Immobilized pH Gradient (IPG) Strips
2.3 Isoelectric Focusing (IEF) of Rehydrated Strips Using IPGphor Device
2.4 10% SDS PAGE Gel
2.5 Semidry Transfer
2.6 Immunoblotting
3 Methods
3.1 Selection of L. intracellularis Proteins that Interact with the IPEC Cells and Their Preparation for 2-DE
3.2 Rehydration of Two 13 Cm pH 4-7 IPG Strips (See Note 9)
3.3 IEF of Rehydrated Strips Using IPGphor
3.4 10% SDS-PAGE Gel, Using Bio-Rad Protean II Xi Cell Apparatus
3.5 Semidry Transfer
3.6 Immunoblotting
3.7 Excising Gel Plugs for Mass Spectrometry
4 Notes
References
Chapter 6: Panproteome Analysis of the Human Antibody Response to Bacterial Vaccines and Challenge
1 Introduction
2 Materials
2.1 Genomic Information
2.2 Data Analysis Software (See Note 2 for Descriptions)
3 Methods
3.1 Select Pathogen
3.2 Identify Conserved and Diverse Proteins
3.3 Classify Proteins in the Core and Panproteome
3.4 Down-Select Proteins from the Core and Panproteome if Necessary
3.5 Statistical Analysis of the Panproteome Antibody Response to Vaccines and Challenge
4 Notes
References
Chapter 7: Low-Energy Electron Irradiation (LEEI) for the Generation of Inactivated Bacterial Vaccines
1 Introduction
2 Materials
2.1 Buffers, Reagents and Media
2.2 Equipment
2.3 Specialized Equipment
3 Methods
3.1 Preparing the Bacterial Solution for Irradiation
3.2 Irradiation in a Small-Scale Experimental Setup
3.3 Irradiation in Bags
3.4 Determining the Antigenicity of the Irradiated Sample by ELISA
4 Notes
References
Chapter 8: Design and Production of Hybrid Antigens for Targeting Integral Outer Membrane Proteins in Gram-Negative Bacteria
1 Introduction
2 Materials
2.1 Autoinduction Medium Components
2.2 Other Media Used for Cultivating Bacteria
2.3 Stock Solutions of Components of Protein Purification Buffers
2.4 Protein Purification Buffers
2.5 Other Materials for Protein Production and Purification
2.6 Materials for Hybrid Antigen Design
2.7 Materials for TBDT Extraction
2.8 ELISA Reagents
3 Methods
3.1 Designing and Preparing Hybrid Antigen Genes
3.2 Expressing Hybrid Protein Antigens in E. coli Using a T7 Expression Vector
3.3 Purification of Soluble Protein Antigens
3.4 Purification of Insoluble Protein Antigens
3.5 Cloning of Tbdt in Fusion with Streptavidin Binding Protein (SBP)
3.6 Preparation of Detergent-Extracted SBP-TBDT Fusion Protein
3.7 Coating Streptavidin ELISA Plates with Detergent-Extracted SBP-TBDT
3.8 Assessing Conformation of TbpA by Its Ability to Bind to Transferrin
3.9 Assessing antibody Titers in Serum Samples from Immunized Animals
4 Notes
References
Chapter 9: Preparation of Trimethyl Chitosan-Based Polyelectrolyte Complexes for Peptide Subunit Vaccine Delivery
1 Introduction
2 Materials
2.1 Synthesis of TMC
2.2 Formulation and Characterization of PECs (LCP-1/Alginate/TMC)
2.3 Equipment
3 Methods
3.1 Synthesis of TMC
3.2 Preparation of PECs (LCP-1/Alginate/TMC)
3.2.1 Prepare the Primary Complexes (LCP-1/Alginate)
3.2.2 Preparation of Ternary PECs (LCP-1/Alginate/TMC)
4 Notes
References
Chapter 10: Multiepitope Fusion Antigen: MEFA, an Epitope- and Structure-Based Vaccinology Platform for Multivalent Vaccine De...
1 Introduction
2 Materials
2.1 Computation and Programs
2.2 A Backbone Protein and Heterogeneous Epitopes of Interest
2.3 MEFA Immunogen Construction, Expression, and Extraction
2.4 Protein Biochemical Property and Immunogenicity Characterization
3 Methods
3.1 Backbone Protein Selection
3.1.1 To Predict Backbone Immunogen Epitopes
3.1.2 To Examine Backbone Protein Stability
3.1.3 To Generate Backbone Protein Model and to Illustrate Protein 3D Images and Epitopes
3.2 Identification of Epitopes from Target Heterogeneous Strains or Virulence Factors
3.2.1 To Predict Epitopes of Heterogeneous Strains or Virulence Factors of Interest
3.2.2 To Identify Protective Epitopes
3.3 MEFA Immunogen Construction
3.3.1 GROMACS to Characterize an MEFA Protein
3.3.2 CHARMM to Characterize MEFA Protein
3.4 MEFA Protein Expression, Extraction, and Immunogenicity Characterization
3.4.1 MEFA Protein Expression and Extraction
3.4.2 MEFA Protein Immunization and MEFA-Specific Antibody Titration
3.4.3 MEFA Protein-Induced Antibody In Vitro and In Vivo Protective Activities
4 Notes
References
Chapter 11: Production, Isolation, and Characterization of Bioengineered Bacterial Extracellular Membrane Vesicles Derived fro...
1 Introduction
2 Materials
2.1 Synthetic Gene Design
2.2 Cloning of Synthetic Gene
2.2.1 Generation of Recombinant DNA
2.2.2 Transformation of Competent Cells
2.2.3 Screening of Recombinant Bacteria
2.3 Conjugative Transfer of Shuttle Vector into Bt
2.4 Assessing Protein Expression and Secretion into BEV
2.4.1 Culture of Bt Transconjugants
2.4.2 Cell Total Protein Extraction
2.4.3 BEV Total Protein Extraction
2.4.4 Protein Western Blotting/Antigen Immunodetection
2.5 Bacteria Medium-Scale Culture
2.5.1 BHIH Bacterial Culture
2.5.2 BDM+ Bacterial Culture
2.5.3 Supernatant Collection
2.6 EV Isolation
2.7 EVs Purification
2.7.1 Routine Purification
2.7.2 High-Resolution Fractionation
2.8 EVs Size and Concentration Analysis
2.9 Antigen Localization with Proteinase K Assay
2.10 Antigen Quantification
3 Methods
3.1 Synthetic Gene Design
3.2 Cloning of Gene of Interest
3.2.1 Generation of Recombinant DNA
3.2.2 Ransformation of Competent Cells
3.2.3 Screening of Cloned Recombinant DNA
3.3 Conjugative Transfer of Shuttle Vector into Bt
3.3.1 Triparental Mating Procedure
3.4 Assessing Protein Expression and Secretion into BEVs
3.4.1 Culture of Bt Transconjugants
3.4.2 Cell Total Protein Extraction
3.4.3 BEV Total Protein Extraction
3.4.4 Protein Western Blotting/Antigen Immunodetection
3.4.5 Medium-Scale Bacterial Culture and Harvesting Conditioned mediaBHIH Bacterial Culture
3.4.6 BDM+ Bacterial Culture
3.4.7 Supernatant Collection
3.5 Isolation of BEVs
3.5.1 Module Rinsing
3.5.2 Sample Concentration
3.5.3 Module Decontamination and Washing
3.6 BEVs Purification
3.6.1 Routine Purification
3.6.2 High-Resolution Size Fractionation
3.7 BEVs Size and Concentration Analysis
3.8 Proteinase K Assay
3.9 Antigen Quantification
4 Notes
References
Chapter 12: Membrane Vesicles Produced by Shewanella vesiculosa HM13 as a Prospective Platform for Secretory Production of Het...
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions and Reagents
2.3 Bacterial Strains, Plasmid, and Culture Media
3 Methods
3.1 Construction of a P49-eGFP Fusion Protein Expression Strain
3.1.1 Construction of pKP49eGFP and Transformation into Conjugal Donor Strain
3.1.2 Conjugal Transfer of pKP49eGFP from E. coli S17-1/ฮปpir into S. vesiculosa HM13-Rifr
3.1.3 Verification of Plasmid Integration into the S. vesiculosa HM13-Rifr Chromosome
3.2 Bacterial Culture for EMV Preparation
3.3 EMV Preparation
3.4 Localization Analysis of P49-Fusion Protein
3.4.1 Preparation of Cellular Protein Samples
3.4.2 Sample Preparation for SDS-PAGE and Western Blotting
3.4.3 SDS-PAGE and Western Blotting
4 Notes
References
Chapter 13: Glycine Induction Method: Effective Production of Immunoactive Bacterial Membrane Vesicles with Low Endotoxin Cont...
1 Introduction
2 Materials
2.1 Induction of E. coli MV Production by Addition of Glycine
2.2 Quantification of MV Components
2.2.1 Quantification of the Amount of Total Protein in MVs
2.2.2 Quantification of the Amount of Total Lipid in MVs
2.2.3 Quantification of Endotoxin in MVs
2.3 Evaluation In Vitro: Cytokine-Inducing Activity of MVs
2.3.1 Addition of MVs to Macrophage-like Cells and Sample Preparation
2.3.2 Quantitative Real-Time PCR
2.3.3 Sandwich ELISA for Cytokines
2.4 Evaluation In Vivo: Mucosal Adjuvanticity of MVs
2.4.1 Intranasal Immunization of Mice
2.4.2 Sample Collection from Mice
2.4.3 ELISA for Evaluation of Mucosal Adjuvanticity of MVs
3 Methods
3.1 Induction of E. coli MV Production by Addition of Glycine
3.2 Quantification of MV Components
3.2.1 Quantification of the Amount of Total Protein in MVs
3.2.2 Quantification of the Amount of Total Lipid in MVs
3.2.3 Quantification of Endotoxin in MVs
3.3 Evaluation In Vitro: Cytokine-Inducing Activity of MVs
3.3.1 Addition of MVs to Macrophage-like Cells and Sample Preparation
3.3.2 Quantitative Real-Time PCR
3.3.3 Cytokine ELISA
3.4 Evaluation In Vivo: Mucosal Adjuvanticity of MVs
3.4.1 Nasal Immunization to Mice
3.4.2 Sample Collection from Mice
3.4.3 ELISA for Evaluation of Mucosal Adjuvanticity of MVs
4 Notes
References
Chapter 14: Methods for Assessment of OMV/GMMA Quality and Stability
1 Introduction
2 Materials
2.1 Dot Blot/Western Blot
2.2 Dynamic Light Scattering (DLS)
2.3 Size Exclusion High-Performance Liquid Chromatography with Multiangle Light Scattering (HPLC-SEC/MALS)
2.4 Nanoparticle Tracking Analysis (NTA)
2.5 SDS-Page
2.6 Micro BCA
2.7 Ultracentrifugation
2.8 Lowry
2.9 Amino Acid Analysis
2.10 High-Pression Anion Exchange Chromatography Coupled with Pulsed Amperometric Detection (HPAEC-PAD) Analysis (Neutral Suga...
2.11 HPAEC-PAD Analysis (Amino Uronic Acid)
2.12 Dische Colorimetric Method
2.13 High-Performance Liquid Chromatography Mass Spectrometry (HPLC-MS) (See Note 3)
2.14 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)
2.15 Competitive-ELISA (cELISA)
2.16 OAg Extraction
2.17 Size Exclusion High-Performance Liquid Chromatography, Derivatization with Semicarbazide (HPLC-SEC SCA)
2.18 Nuclear Magnetic Resonance (NMR)
2.19 Hestrin Colorimetric Method
3 Methods
3.1 Dot Blot/Western Blot
3.2 Dynamic Light Scattering
3.3 HPLC-SEC/MALS
3.4 Nanoparticle Tracking Analysis (NTA)
3.5 SDS-Page
3.6 Micro BCA
3.7 Ultracentrifugation
3.8 Lowry
3.9 Amino Acid Analysis
3.10 Total Sugar Quantification
3.11 HPAEC-PAD (Neutral Sugars)
3.12 HPAEC-PAD (Amino Uronic Acid)
3.13 Dische Colorimetric Method
3.14 HPLC-MS
3.15 MALDI-TOF MS
3.16 cELISA
3.17 OAg Extraction
3.18 HPLC-SEC SCA
3.19 NMR
3.20 Hestrin Colorimetric Method
4 Notes
References
Chapter 15: Production of Vaccines Using Biological Conjugation
1 Introduction
2 Materials
2.1 Cell Growth
2.2 Cell Harvesting and Lysis
2.3 AKTA High-Performance Liquid Chromatography
2.4 HisTrap Purification
2.5 Resource S Purification
2.6 PD-10 Desalting and Endotoxin Removal
2.7 SDS-Page
2.8 Western Blot
2.9 Coomassie Staining
3 Methods
3.1 Cell Growth and Expression
3.2 Cell Lysis
3.3 Chromatography Purification with HisTrap Columns
3.4 SDS-Page
3.5 Western Blot
3.6 Coomassie Stain
3.7 Desalting and Endotoxin Removal
3.8 Cation Exchange Chromatography
3.9 Buffer Exchange and Storage
4 Notes
References
Chapter 16: Immunological Assessment of Lung Responses to Inhalational Lipoprotein Vaccines Against Bacterial Pathogens
1 Introduction
2 Materials
2.1 Leukocyte Preparation
2.2 Antigen Restimulation of Leukocyte Suspensions and Flow Cytometry
2.3 ELISpot
2.4 ELISA
3 Methods
3.1 Isolation of BAL and Lung Leukocytes from Vaccinated Mice
3.1.1 Bronchoalveolar Lavage Collection and Cell Isolation
3.1.2 Lung Leukocytes
3.2 Assessment of Antigen-Specific T-cell Responses Via Ex vivo Antigen Restimulation, Intracellular Cytokine Immunostaining a...
3.2.1 Restimulation of Antigen-Specific T-cells in Ex vivo Culture
3.2.2 Immunostaining Cells for Detection of Intracellular Cytokine
3.3 IFNฮณ ELISpot
3.3.1 Preparation and Coating of the ELISpot Plate
3.3.2 Plating of Leukocytes for Antigen Restimulation
3.3.3 Staining and Developing the ELISpot Plate
3.4 Sustained Leukocyte Restimulation for Recall of Memory Antigen-Specific T-cell Responses
3.5 Quantitation of Vaccine-Induced Antigen-Specific Antibodies by ELISA
4 Notes
References
Chapter 17: Determination of Maternal and Infant Immune Responses to Pertussis Vaccination in Pregnancy
1 Introduction
2 Materials
2.1 Patient Samples
2.2 Collection and Processing of Whole Blood
2.3 Heat-Killed B. pertussis
2.4 In Vitro Whole Blood Stimulation Assay
2.5 Detection of Cytokines by Multiplex and Singleplex Assays
2.6 Contribution of the Plasma Compartment in Cord Blood Cytokine Responses to B. pertussis
3 Methods
3.1 Preparation of Heat-Killed B. pertussis and LPS Stocks
3.2 Bulk Preparation of B. pertussis Stimulation Plates
3.3 Processing of Maternal, Cord, and Infant Whole Blood
3.4 In Vitro B. pertussis Stimulation Assay
3.5 Meso Scale Discovery (MSD) Multiplex Cytokine Assay
3.6 IL-8 ELISA
3.7 Contribution of the Plasma Compartment in Cord Blood Cytokine Responses to B. pertussis
4 Notes
References
Chapter 18: Generation of a Universal Human Complement Source by Large-Scale Depletion of IgG and IgM from Pooled Human Plasma
1 Introduction
2 Materials
2.1 Plasma and Blood Plasma Preparation
2.2 Sanitization of Chromatography System and Columns (See Note 3)
2.3 Chromatography Buffers (See Note 3)
2.4 Chromatography Columns
2.5 Chromatography Systems
2.6 Concentration of Plasma, Complement, and C1q
2.7 Ancillary Items
2.8 QC Testing-Bioburden
2.9 QC Testing-Presence of Ig and C1q
2.10 QC Testing-Complement Component Activity
3 Method
3.1 Plasma Preparation
3.2 Column Setup and Sanitization
3.2.1 Column Preparation
3.2.2 Sanitization of Chromatography Systems with Sodium Hydroxide (See Notes 11 and 12)
3.2.3 Sanitization of Columns at 4 C with 70% Ethanol (See Note 14)
3.3 IgG and IgM Depletion of Plasma
3.3.1 Equilibration of the Chromatography Systems and Columns at 4 C
3.3.2 Loading the Plasma onto the Columns
3.4 C1q Elution, Concentration of Components and Reconstitution
3.4.1 Elution of C1q at 22 C with Salt Gradient (See Note 16)
3.4.2 Elution of IgG and IgM for Reuse of Column (See Note 19)
3.4.3 Concentration of Plasma and C1q and Recovery from Dialysis Tubing
3.4.4 Reconstitution of Complement with C1q and Aliquoting for Storage
3.5 Batch Quality Testing
3.5.1 SDS-PAGE and Western Blotting
3.5.2 Bioburden Check
3.5.3 Confirmation of Complement Activity
4 Notes
References
Chapter 19: Assessment of Serum Bactericidal and Opsonophagocytic Activity of Antibodies to Gonococcal Vaccine Targets
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Assay Media
3.2 Preparation of Bacterial Inoculum
3.3 Heat Inactivation of Immune Sera
3.4 Preparation of Serum for the Complement Source
3.5 Preparation of Polymorphonuclear Cells
3.6 SBA Assay
3.7 OPA Assay
4 Notes
References
Chapter 20: Opsonophagocytic Killing Assay to Measure Anti-Group A Streptococcus Antibody Functionality in Human Serum
1 Introduction
2 Materials
3 Methods
3.1 HL-60 Master Stock Propagation from Master Cell Bank Stock
3.2 HL-60 Working Stock Preparation from Master Stock
3.3 Differentiation of HL-60 Cells
3.4 Bacterial Master Stock Preparation and Maintenance
3.4.1 From Clinical Swabs
3.4.2 From Plated Bacteria
3.5 Bacterial Expansion
3.6 Human Serum Sample Preparation
3.7 Preparing Working Aliquots of Baby Rabbit Complement (BRC)
3.8 Preparation of Differentiated HL-60 Cells (for Use in the Assay)
3.9 Preparation of Bacteria (for Use in the Assay)
3.10 Procedure for Determining Optimal Dilution of Bacteria
3.10.1 Optimal Dilution Experiment 1
3.10.2 Optimal Dilution Experiment 2
3.11 OPKA Procedure
3.12 Data Analysis
3.12.1 Limits of Detection
3.12.2 Tentative Plate Acceptance Criteria
3.12.3 Tentative Sample Acceptance Criteria
4 Notes
References
Chapter 21: Neisseria lactamica Controlled Human Infection Model
1 Introduction
2 Materials
2.1 Dilution of N. lactamica Stock Vials to 105 CFU/ml for Intranasal Inoculation
2.2 Intranasal Inoculation with 105 CFU N. lactamica
2.3 Taking and Processing an Oropharyngeal Throat Swab to Determine Volunteer N. lactamica Colonization Status
2.4 Taking and Processing a Nasal Wash Sample to Determine Volunteer N. lactamica Colonization Status
2.5 Identification of N. lactamica Colonies
2.6 Identification of N. lactamica Colonies as Strain Y92-1009 Using PCR
2.6.1 Preparation of PCR Master Mix
2.6.2 Performance of Y92-1009-Specific PCR
2.6.3 Analysis of PCR Products Using Agarose Gel Electrophoresis
3 Methods
3.1 Dilution of N. lactamica Stock Vials to 105 CFU/ml for Intranasal Inoculation
3.2 Intranasal Inoculation with 105 CFU N. lactamica
3.3 Taking and Processing Oropharyngeal Throat Swabs to Determine Volunteer N. lactamica Colonization Status
3.4 Taking and Processing Nasal Wash Samples to Determine Volunteer N. lactamica Colonization Density
3.5 Identification of N. lactamica Colonies from Throat Swab and Nasal Wash Cultures
3.6 Identification of N. lactamica Colonies as Strain Y92-1009 Using PCR
3.6.1 Preparation of PCR Master Mix
3.6.2 Performance of Y92-1009-Specific PCR
4 Notes
References
Chapter 22: Analyzing Macrophage Infection at the Organ Level
1 Introduction
2 Materials
2.1 Bacterial Strains and Culture Conditions
2.2 Murine Infection Model
2.3 Ex Vivo Porcine Spleen Perfusion
2.4 Sample Freezing and Sectioning
2.5 Whole Slide Scanning
2.6 Image Analysis
3 Methods
3.1 Bacterial Strains and Culture Conditions
3.1.1 Broth
3.1.2 Blood Agar Base (BAB) Culture Plates + 3% v/v Horse Blood
3.1.3 Bacterial Infection Stocks
3.2 Murine Infection Model
3.2.1 Infection Dose
3.2.2 Intravenous Route of Infection
3.2.3 Monitor Signs of Disease and Euthanasia
3.2.4 Organ Recovery and Bacterial Enumeration
3.3 Ex Vivo Porcine Liver and Spleen Coperfusion Model
3.3.1 Set up of the Ex Vivo Perfusion Circuit
3.3.2 Organ Retrieval
3.3.3 Ex Vivo Perfusion and Bacterial Infection
3.4 Sample Preparation
3.4.1 Sample Freezing
3.4.2 Sample Sectioning
3.4.3 Indirect Immunohistochemistry Staining
3.5 Sample Microscopy and Image Analysis
3.5.1 Fluorescence Whole-Slide Scanning
3.5.2 inForm Image Analysis
3.5.3 Fiji Image Analysis
4 Notes
References
Chapter 23: Multicolor Flow Cytometry and High-Dimensional Data Analysis to Probe Complex Questions in Vaccinology
1 Introduction
2 Materials
2.1 Equipment
2.2 Software
2.3 Reagents
3 Methods
3.1 Preparation of Reagents
3.2 Preparation of PBMC for Multidimensional Flow Cytometry Analysis
3.3 Acquiring Events on the Flow Cytometer
3.4 Data Analysis
3.4.1 Preliminary Analysis
3.4.2 Data Down Sampling and Concatenation
3.4.3 Running the t-SNE
3.4.4 FlowSOM
4 Notes
References
Index