Preface
Contents
Contributors
The Simplest Protocol for Rapid and Long-Term Culture of Primary Epidermal Keratinocytes from Human and Mouse
1 Introduction
2 Materials
2.1 Mouse
2.2 Cells
2.3 Cell Culture Reagents and Media
2.4 Other Reagents and Chemicals
2.5 Instruments and Supplies
2.6 Preparation of Cell Culture Media and Freezing Media
2.6.1 CnT-PR Medium
2.6.2 J2 Medium
2.6.3 FAD Medium
2.6.4 Complete FAD (cFAD) Medium
2.6.5 hPA Medium
2.6.6 Freezing Medium
2.7 Preparation of Cell Culture Reagents
3 Methods
3.1 Collection of Crude Primary Epidermal Cells from Mouse Skin
3.2 Enrichment and Expansion of Mouse Primary Epidermal Keratinocytes in Serum-Free Media
3.3 Preparation of Human Epidermal Keratinocyte Stocks
3.4 Preparation of Feeder Cells for Co-culture with Epidermal Keratinocytes
3.4.1 Preparation of Feeder Cells
3.4.2 Culture, Passage, and Storage of 3T3-J2 Cells and hDFs
3.4.3 Culture, Passage, and Storage of hPAs
3.5 Promotion of Human and Mouse Primary Epidermal Keratinocyte Expansion in Co-culture with Feeder Cells Using a TGF-ฮฒ Signal...
3.5.1 Preparation of Feeder Cell Layers
3.5.2 Setting Up Co-culture
3.5.3 Visualization of Epidermal Keratinocyte Clones in Feeder Cell Co-culture by Rhodamine B Staining
3.5.4 Immunofluorescence Staining of p63 and CK5 in Human Epidermal Keratinocyte Clones in Feeder Cell Co-culture
4 Notes
References
Dermal-Epidermal Separation by Heat
1 Introduction
2 Materials
3 Methods
4 Notes
References
Dermal-Epidermal Separation by Enzyme
1 Introduction
2 Materials
3 Methods
4 Notes
References
Dermal-Epidermal Separation by Chemical
1 Introduction
2 Materials
3 Methods
4 Note
References
Isolation and Characterization of Extracellular Vesicles from Keratinocyte Cultures
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Extracellular Vesicle Isolation Using Size-Exclusion Chromatography
2.3 Nanoparticle Tracking Analysis
2.4 Transmission Electron Microscopy
2.5 Western Blot
3 Methods
3.1 Expansion of Cells and Production of Conditioned Media
3.2 Concentration of Conditioned Media and EV Isolation Using Size-Exclusion Chromatography
3.3 Nanoparticle Tracking Analysis (NTA)
3.4 Transmission Electron Microscopy
3.5 Western Blot
4 Notes
References
Competitive Repopulation Assay of Long-Term Epidermal Stem Cell Regeneration Potential
1 Introduction
2 Materials
2.1 Mice
2.2 Reagents and Solutions
2.3 Tools and Tubes
3 Methods
3.1 Isolation of Adult Skin Keratinocytes and Neonatal Skin Fibroblasts: Storage and Retrieval of Frozen Stocks
3.2 Chamber Implantation and Cell Transplantation
3.3 Keratinocyte Isolation and Flow Cytometry Analysis of the myrAkt-HA Versus Competitor WT-GFP Long-Term Repopulating Kerati...
3.3.1 Isolation of Keratinocytes from Grafts
3.3.2 Preparation of the Control and Labelled Sample Tubes for Flow Cytometry Analysis
3.3.3 Overall Guidelines for the Flow Cytometry Analysis of the Samples
4 Notes
References
Quantification of Melanosome Transfer Using Immunofluorescence Microscopy and Automated Image Analysis
1 Introduction
2 Materials
2.1 Cells for Co-culture
2.2 Reagents for Co-culture
2.3 Reagents for Immunofluorescence
2.4 Instruments
2.5 Software for Analysis
3 Methods
3.1 UVB Treatment and Co-culturing Melanocytes and Keratinocytes
3.2 Immunofluorescence Staining
3.3 Image Acquisition
3.4 Image Quantitation
4 Notes
References
Contribution of Immunohistochemistry in Revealing S100A7/JAB1 Colocalization in Psoriatic Epidermal Keratinocyte
1 Introduction
2 Materials
2.1 S100A7/JAB1 Double Immunohistochemical Staining in Formalin-Fixed and Paraffin-Embedded Skin Tissue
2.2 S100A7/JAB1 Double Immunocytochemical Staining in Normal Human Keratinocyte Cell Culture
3 Methods
3.1 S100A7/JAB1 Double Immunohistochemical Staining (Sequential and Indirect Method) in Formalin-Fixed and Paraffin-Embedded S...
3.2 S100A7/JAB1 Double Immunocytochemical Staining (Sequential and Indirect Method) in Normal Human Keratinocyte Cell Culture
3.3 Semiquantitative Immunostaining Evaluation
4 Notes
References
Digital Quantification of Epidermal Protein Expression in Paraffin-Embedded Tissue Using Immunohistochemistry
1 Introduction
2 Materials
2.1 Primary Antibody Preparation
2.2 Sample Processing
2.3 Slide Staining
2.4 Slide Scanning
2.5 Software
3 Methods
3.1 Primary Antibody Preparation
3.2 Slide Preparation
3.3 Staining
3.4 Automated Immunohistochemistry Staining Protocol
3.5 Slide Scanning
3.6 Convert Slide Scanned Images to Standard Format
3.7 Separate Epidermis from Complete Slide
3.8 Measure Chromophore Intensity
4 Notes
References
Keratinocyte Differentiation by Flow Cytometry
1 Introduction
2 Materials
2.1 Fixing and Permeabilising Cells
2.1.1 Keratins
2.1.2 Involucrin
2.2 Primary Antibody Staining
2.2.1 Keratins
2.2.2 Involucrin
2.3 Secondary Antibody Staining
2.3.1 Keratins
2.3.2 Involucrin
2.4 Running Flow Cytometry
2.5 Equipment
3 Methods
3.1 Fixing and Permeabilising Cells
3.1.1 Keratins
3.1.2 Involucrin
3.2 Primary Antibody Staining
3.2.1 Keratins
3.2.2 Involucrin
3.3 Secondary Antibody Staining
3.3.1 Keratins
3.3.2 Involucrin
3.4 Running Flow Cytometry
3.5 Analysis
4 Notes
References
In Silico and In Vitro Considerations of Keratinocyte Nuclear Receptor Protein Structural Order for Improving Experimental Ana
1 Introduction
2 Materials
2.1 Bacteria and Bacterial Expression Vectors
2.2 Reagents
3 Methods
3.1 In Silico Assessment for Predicting Disorder and Unique IDP/IDR Characteristics
3.2 Recombinant Design and Expression of IDP or IDR
3.3 Methods in Recombinant IDP Purification
3.4 Methods for Biophysical Analysis
3.4.1 Limited Proteolysis
3.4.2 Proteasome Methods
4 Notes
References
Genetic Modification of Human Primary Keratinocytes by Lentiviral Vectors
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 Keratinocyte Culture in High-Calcium Medium (1.2 mM; Rheinwald FAD Medium; Complete FAD)
2.1.2 Keratinocyte Culture in Low-Calcium Media (<0.1 mM)
2.2 Lentiviral Production and Infection
2.2.1 Producer Cells
2.2.2 Transfection Reagents
2.2.3 Lentiviral Plasmids
2.2.4 Infection Reagents
2.3 Antibiotics
3 Methods
3.1 Lentiviral Infection in Low-Calcium Medium
3.1.1 Lentiviral Production
3.1.2 Harvest and Plate Keratinocytes
3.1.3 Keratinocyte Infection
3.1.4 Checking Infection and Stable Selection
3.2 Lentiviral Infection in High-Calcium Medium
3.2.1 Lentiviral Production
3.2.2 Harvest and Plate Keratinocytes
3.2.3 Lentiviral Infection
3.2.4 Checking Infection and Stable Selection
4 Notes
References
Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9
1 Introduction
2 Materials and Equipment
2.1 General Lab Consumables and Equipment
2.2 Generation of Puromycin-Resistant 3T3-J2 Feeder Cells
2.2.1 Lentivirus Production for Transduction of 3T3-J2 Cells
2.2.2 Culturing and Transduction of 3T3-J2 Cells
2.2.3 Equipment
2.3 Generation of Mitotically Inactivated 3T3-J2 Feeder Cells
2.3.1 Generation of Mitotically Inactivated 3T3-J2 Feeder Cells by Mitomycin C Treatment
2.3.2 Generation of Mitotically Inactivated 3T3-J2 Feeder Cells by ฮณ-Irradiation
2.3.3 Equipment
2.4 Isolation of Human Primary Keratinocytes (HPKs)
2.4.1 Equipment
2.5 Generation of CRISPR/Cas9-Mediated Knockout HPKs
2.5.1 Lentivirus Production for Transducing HPKs
2.5.2 Lentiviral Transduction and Selection of HPKs Co-cultured with 3T3-J2 Cells
2.5.3 Expansion of CRISPR/Cas9 Knockout HPKs
2.5.4 Equipment
2.6 Seeding CRISPR/Cas9 Knockout HPKs for Experiments
2.6.1 Equipment
3 Methods
3.1 Generation of Puromycin-Resistant 3T3-J2 Feeder Cells
3.1.1 Lentivirus Production for Transduction of 3T3-J2 Cells
3.1.2 Culturing and Transduction of 3T3-J2 Cells
3.2 Generation of Mitotically Inactivated 3T3-J2 Feeder Cells
3.2.1 Generation of Mitotically Inactivated 3T3-J2 Feeder Cells by Mitomycin C Treatment
3.2.2 Generation of Mitotically Inactivated 3T3-J2 Feeder Cells by ฮณ-Irradiation
3.3 Isolation of Human Primary Keratinocytes
3.4 Generation of CRISPR/Cas9-Mediated Knockout HPKs
3.4.1 Lentivirus Production for Transduction of HPKs
3.4.2 Lentiviral Transduction and Selection of HPKs Co-cultured with 3T3-J2 Cells
3.4.3 Expansion of CRISPR/Cas9 Knockout HPKs
3.5 Seeding CRISPR/Cas9 Knockout HPKs for Experiments
4 Notes
References
In Vitro Wound Healing Assays to Investigate Epidermal Migration
1 Introduction
2 Materials
2.1 Preparation of Coated Plates
2.2 Preparation of Conditioned Media from HEK-293T Cells
2.3 Scratch Assay
2.4 Imaging and Quantification of Epidermal Wound Healing
3 Methods
3.1 Preparation of Coated Plates
3.2 Preparation of HEK-293T-Conditioned Media
3.3 Epidermal Cell Scratch Assay
3.4 Analysis of Epidermal Migration in Scratch Assay
4 Notes
References
Iterative Three-Dimensional Epidermis Bioengineering and Xenografting to Assess Long-Term Regenerative Potential in Human Kera
1 Introduction
2 Materials
2.1 Cellular Material
2.2 Recipient Mice
2.3 Equipment
2.3.1 Flow Cytometry
2.3.2 Xenografting
2.3.3 Histology
2.4 Reagents and Products
2.4.1 Keratinocyte Culture Medium
2.4.2 Skin Substitute Bioengineering and Xenografting
2.4.3 Graft Dissociation
2.4.4 Flow Cytometry Cell Sorting
2.4.5 Anesthesia
3 Methods
3.1 Description of the Assay Principle
3.2 Primary Skin Substitute Bioengineering
3.3 Xenografting of Primary Skin Substitutes onto Recipient Mice
3.4 Dissection of Primary Grafts and Isolation of Human Keratinocytes
3.5 Secondary Skin Substitute Bioengineering
3.6 Xenografting of Secondary Epidermis Substitutes onto Recipient Mice
4 Notes
References
Generation of a Full-Thickness Human Skin Equivalent on an Immunodeficient Mouse
Abbreviations
1 Introduction
2 Materials
2.1 Cell Lines and Culture
2.2 Grafting Procedure
2.3 Graft Harvesting
2.4 Equipment
3 Methods
3.1 Culturing Keratinocytes
3.1.1 Coating Tissue Culture Dishes with Collagen
3.1.2 Thawing and Maintaining Keratinocytes
3.1.3 Keratinocyte Subculture
3.2 Culturing Fibroblasts
3.2.1 Thawing and Maintaining Fibroblasts
3.2.2 Fibroblast Subculture
3.3 Harvesting Cells for Grafting
3.3.1 Collecting Keratinocytes
3.3.2 Collecting Fibroblasts
3.4 Chamber Insertion
3.5 Chamber Removal
3.6 Graft Harvesting
4 Notes
References
Biotin Identification Proteomics in Three-Dimensional Organotypic Human Skin Cultures
1 Introduction
2 Materials
2.1 Biotin Stock Solution
2.2 Check Keratinocytes for Biotinylating Activity
2.3 Urea Sample Buffer
2.4 Amido Black Protein Assay
2.5 Streptavidin Precipitation
2.6 Washing Beads After Precipitation
2.7 Isolation of Peptides for Mass Spectrometry
2.8 Check Samples for Mass Spectrometry
3 Methods
3.1 Check Cells for Biotinylating Activity
3.1.1 Biotinylating Activity in 2D Cultures
3.1.2 Biotinylating Activity in 3D Cultures
3.2 Cell Treatment and Harvest for Mass Spectrometry
3.3 Amido Black Protein Assay (See Note 10)
3.4 Streptavidin Precipitation (See Note 11)
3.5 Washing Beads After Precipitation
3.6 Isolation of Peptides for Mass Spectrometry
3.7 Check Samples for Mass Spectrometry
4 Notes
References
Information and Statistical Analysis Pipeline for High-Throughput RNA Sequencing Data
1 Introduction
2 Materials
2.1 The Data Format of Sequencing Reads
2.2 Data Acquisition from Public Databases
3 Methods
3.1 Quality Check and Trimming
3.2 Mapping and Counting
3.3 Normalization
3.4 Classification and Clustering
3.5 Differentially Expressed Gene (DEG) Analysis
3.6 Enrichment Analysis
3.7 Visualization
4 Notes
4.1 Computer Programming Languages
4.2 Reproducibility, Open Source, and Cloud Service
4.3 Data Generation by Long-Read Sequencers
4.4 Batch Effect Correction
4.5 Independent Filtering
4.6 Single-Cell RNA Sequencing
References
Cell-Extracellular Matrix Adhesion Assay
1 Introduction
2 Materials
3 Methods
3.1 HaCaT Cell Culture
3.2 Preparation of the Extracellular Matrix-Coated Plates
3.3 Adhesion Assay
4 Notes
References
Methods for Analysis of Keratinocyte Migration
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.3 Solution
3 Methods
3.1 Culture of Primary Mouse Keratinocytes
3.2 Coating of Culture Plates
3.3 Passaging Keratinocytes
3.4 Scratch Wounding Assay of Keratinocyte
3.5 Cell Motility Assay
4 Notes
References
Scratch Wound Healing Assay
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture Preparation
3.2 Scratch Wound Assay
3.3 Incubation Time
3.4 Data Acquisition
3.5 Data Analysis
4 Notes
References
Photoprotective Activity Assay Toward Ultraviolet B in Human Keratinocytes
1 Introduction
2 Materials
3 Methods
3.1 HaCaT Cell Culture
3.2 Photoprotective Activity Assay
4 Notes
References
Photodynamic Therapy Assay
1 Introduction
2 Materials
3 Methods
3.1 Cell Culture
3.2 Photodynamic Therapy Assay
4 Notes
References
A Method to Prepare Claudin-Modulating Recombinant Proteins
1 Introduction
2 Materials
2.1 Bacterial Strains
2.2 Bacteria Culture Media
2.3 Plasmids
2.4 Protein Purification Materials
2.5 Cell Lines
2.6 Cell Culture Medium
2.7 Reagents for Flow Cytometric Analysis
2.8 Equipment for Measuring TEER
3 Methods
3.1 Preparation of Claudin-Modulating Recombinant Proteins
3.2 Flow Cytometric Analysis
3.3 Assay of Cell Monolayer Tight Junctions
4 Notes
References
Human Fetal Skin Fibroblast Isolation and Expansion for Clinical Application
Abbreviations
1 Introduction
2 Materials
2.1 Human Fetus Procurement
2.2 Fetal Skin Harvesting, Fibroblast Isolation, Culture, Subculture, Cryopreservation, and Banking
3 Methods
3.1 Controls Prior to Skin Tissue Processing
3.2 Fetal Skin Preparation
3.3 Fibroblast Isolation from Skin Tissue
3.4 Fetal Skin Fibroblast Culture and Subcultures
3.5 Characterization of Cultured Fetal Skin Fibroblasts
3.6 Cryopreservation
3.7 Banking and Releasing for Clinical Transplantation
4 Notes
References
Ciliated Epithelial Cell Differentiation at Air-Liquid Interface Using Commercially Available Culture Media
1 Introduction
2 Materials
2.1 Nasal Airway Brushing
2.2 3T3-J2 Feeder Culture
2.3 Nasal Epithelial Basal Cell Culture in 3T3-J2 Co-culture
2.4 Differentiation of Nasal Epithelial Cells at Air-Liquid Interface (ALI)
2.5 Immunofluorescence Staining of ALI Cultures
2.6 Western Blotting
2.7 Flow Cytometry (FACS)
3 Methods
3.1 Preparation of 3T3-J2 Feeder Layers
3.2 Isolation and Expansion of Human Nasal Epithelial Cells
3.2.1 Cell Seeding from Brushings
3.2.2 Maintenance of Human Nasal Basal Epithelial Cells
3.3 Air-Liquid Interface Culture of Human Nasal Epithelial Cells
3.3.1 Preparation of Collagen-Coated Transwell Inserts
3.3.2 Differentiation of Nasal Epithelial Cells at Air-Liquid Interface (ALI)
3.4 Transepithelial Electrical Resistance (TEER) Measurements
3.5 Ciliary Beat Frequency Analysis
3.6 Immunofluorescence
3.7 Western Blotting
3.8 Flow Cytometry
3.9 Comparison of Commercially Available Medium
4 Notes
References
Correction to: Isolation and Characterization of Extracellular Vesicles from Keratinocyte Cultures
Index