Molecular Dermatology: Methods and Protocols (Methods in Molecular Biology, 2154)

Preface
Contents
Contributors
Chapter 1: Isolation of Epidermal Keratinocytes from Human Skin: The Scratch-Wound Assay for Assessment of Epidermal Keratinoc...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Human Skin Tissue Processing and Primary EK Extraction
		3.2 Culture and Passage of Primary Human EKs
		3.3 The Scratch-Wound Healing Assay
		3.4 Analysis and Interpretation of the Data
	4 Notes
	References
Chapter 2: Isolation of Different Dermal Fibroblast Populations from the Skin and the Hair Follicle
	1 Introduction
	2 Materials
		2.1 Tissue Preparation
		2.2 Cell Culture Maintenance
		2.3 Equipment
	3 Methods
		3.1 Tissue Preparation
		3.2 Papillary and Reticular Dermal Fibroblast Isolation
		3.3 Hair Follicle Dermal Sheath and Dermal Papilla Isolation
		3.4 Cell Culture Maintenance and Passage
	4 Notes
	References
Chapter 3: Isolation of Epidermal and Hair Follicle Melanocytes
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
	3 Methods
		3.1 Isolation of Epidermal Melanocytes
		3.2 Isolation of Hair Follicle Melanocytes
	4 Notes
	References
Chapter 4: Isolation and Culture of Human Skin Mast Cells
	1 Introduction
	2 Materials
		2.1 Buffer and Media
		2.2 Antibodies/Microbeads for Flow Cytometry and Magnetic Cell Enrichment
		2.3 Additional Equipment
	3 Methods
		3.1 Tissue Digestion
		3.2 Magnetic Cell Enrichment (MACS)
			3.2.1 Flow Cytometry
			3.2.2 Long-Term Culture
	4 Notes
	References
Chapter 5: Organotypic 3D Skin Models: Human Epidermal Equivalent Cultures from Primary Keratinocytes and Immortalized Keratin...
	1 Introduction
	2 Materials
		2.1 Primary Adult Keratinocyte Isolation
		2.2 3D Epidermal Equivalents
	3 Methods
		3.1 Primary Adult Keratinocyte Isolation (T: Troubleshooting)
		3.2 3D Human Epidermal Equivalents (HEEs)
		3.3 Readout Analyses
			3.3.1 Proliferation Analysis
			3.3.2 Dye Permeation
				Transepidermal Water Loss
				Transepithelial Electrical Resistance
				Tissue Morphology, Gene and Protein Expression
	4 Notes
	5 Troubleshooting
	References
Chapter 6: Purification of Extracellular Microvesicles Secreted by Dermal Fibroblasts
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 sEV Purification
		2.3 Tube Washes and Sterilization
		2.4 Equipment
	3 Methods
		3.1 Depletion of Serum EVs
		3.2 Cell Amplification
		3.3 EV Purification
		3.4 Tube Cleaning and Sterilization
	4 Notes
	References
Chapter 7: A Method to Investigate the Epidermal Permeability Barrier In Vitro
	1 Introduction
		1.1 Overview of the Epidermal Permeability Barrier
		1.2 Using Cultured Keratinocytes for Investigating the Epidermal Permeability Barrier
		1.3 Features Needed in Cultured Keratinocytes for Investigating Epidermal Permeability Barrier
		1.4 Submerged Cultured Keratinocytes to Recapture Proliferating Keratinocytes and Early and Late Stages of Differentiated Kera...
		1.5 Organotypic Epidermal Equivalents
		1.6 Evaluation of Epidermal Permeability Barrier Constituents
		1.7 Thin-Layer Chromatography
		1.8 Studies of Physiological Epidermal Barrier Function
			1.8.1 TEER Assay
			1.8.2 Epidermal Permeability Barrier Recovery Assay by TEER Measurement
			1.8.3 TEWL Measurement
	2 Materials
		2.1 Preparation of Primary Cultured Human Keratinocytes
		2.2 Organotypic Epidermal Equivalents
		2.3 Lipid Extraction from Cultured Cells and Saponification
		2.4 Thin-Layer Chromatography
		2.5 Equipment
	3 Method
		3.1 Primary Culture of Human Keratinocytes
			3.1.1 Skin
		3.2 Submerged Cultured Keratinocytes to Recapture Proliferating Keratinocytes and Early and Late Stages of Differentiated Kera...
		3.3 Organotypic Epidermal Equivalents
		3.4 Evaluation of Epidermal Permeability Barrier Constituents
			3.4.1 Unbound Lipid Extraction from Cultured Cells
			3.4.2 Thin-Layer Chromatography
				Separation of Triglyceride, Free Fatty Acid, Cholesterol, Ceramide, and Glucosylceramide
				Separation of Glucosylceramide
				Development for Separation of Ceramide
				Separation for Phospholipids
				Visualizing Lipids on TLC Plate
			3.4.3 Corneocyte Lipid Envelope (CLE) Analysis
		3.5 Studies of Physiological Epidermal Barrier Function
			3.5.1 TEER Assay
			3.5.2 Epidermal Permeability Barrier Recovery Assay by TEER Measurement
			3.5.3 TEWL Measurement
	4 Notes
	References
Chapter 8: Isolating Dermal Papilla Cells from Human Hair Follicles Using Microdissection and Enzyme Digestion
	1 Introduction
	2 Materials
		2.1 Microdissection
		2.2 Papilla Adherence and Growth
	3 Methods
		3.1 Microdissection of Hair Follicles
			3.1.1 End Bulb Transection
			3.1.2 Inversion of End Bulbs
			3.1.3 Separating the DP from the Inverted End Bulb
		3.2 Adherence of the DP
			3.2.1 Attachment Using Needles
			3.2.2 Enzyme Digestion
	4 Notes
	References
Chapter 9: Methods to Study Human Hair Follicle Growth Ex Vivo: Human Microdissected Hair Follicle and Human Full Thickness Sk...
	1 Introduction
	2 Materials
		2.1 Instruments and Tools
		2.2 William´s E Culture Medium (WCM)
		2.3 Human Samples
	3 Methods
		3.1 Scalp Skin Preparation for HF Microdissection
		3.2 Microdissection of Amputated HFs from Scalp Skin
		3.3 Microdissection of Full-Length HFs from Scalp Skin
		3.4 Microdissection of Amputated HFs Ex Vivo from FUEs
		3.5 Microdissection of Full-Length HFs Ex Vivo from FUEs
		3.6 Plating of Amputated or Full-Length Microdissected HFs
		3.7 Initiation of the Culture of Amputated or Full-Length Microdissected HFs Ex Vivo
		3.8 Continuation and Termination of the Culture of Amputated or Full-Length Microdissected HFs Ex Vivo
		3.9 Full-Thickness Human Scalp Skin Organ Culture with Terminal Hair Follicles Ex Vivo
	4 Notes
	References
Chapter 10: Nonsurgical Induction of Alopecia Areata in C3H/HeJ Mice via Adoptive Transfer of Cultured Lymphoid Cells
	1 Introduction
	2 Materials
		2.1 Reagents and Animals
		2.2 Equipment
		2.3 Reagent Setup
	3 Methods
		3.1 Isolation of Skin-Draining Lymph Nodes
		3.2 Separation of Lymph Node Cells (LNCs) into Single Cell Suspension
		3.3 Activation and Expansion of LNCs
		3.4 Preparation of Expanded LNCs for Injection
		3.5 Adoptive Transfer of LNCs into Naïve C3H/HeJ Mice by Intradermal Injection
		3.6 Development of Alopecia Areata
		3.7 Anticipated Results
			3.7.1 Single Cell Suspension and LNC Expansion
			3.7.2 The Progression of Alopecia Areata in Cell-Injected Mice
	4 Notes
	References
Chapter 11: Collapse and Restoration of Hair Follicle Immune Privilege Ex Vivo: A Model for Alopecia Areata
	1 Introduction
	2 Materials
		2.1 Instruments and Tools
		2.2 Tissue Preparation
	3 Methods
		3.1 Experimental Induction of the Collapse of Immune Privilege in Human Scalp Hair Follicles Ex Vivo
		3.2 Assay for Evaluation of Prevention of Hair Follicle Immune Privilege Collapse
		3.3 Assay for Evaluation of Restoration of Hair Follicle Immune Privilege Collapse
	4 Notes
	References
Chapter 12: Experimentally Induced Epithelial-Mesenchymal Transition of Human Hair Follicle Stem Cells as a Model of Scarring ...
	1 Introduction
	2 Materials
		2.1 Instruments and Tools
		2.2 Human Microdissected Full-Length Anagen VI Scalp Hair Follicles
		2.3 Reagents
	3 Methods
		3.1 Experimental EMT-Induction in Human Microdissected Full-Length Anagen VI Scalp Hair Follicles Ex Vivo
		3.2 Prevention EMT Assay
		3.3 Restoration EMT Assay
	4 Notes
	References
Chapter 13: Generation of Hair Follicle Germs In Vitro Using Human Postnatal Skin Cells
	1 Introduction
	2 Materials
		2.1 Human Tissue Samples
		2.2 Equipment
		2.3 Human Dermal Papilla Isolation and Culturing
		2.4 Human Primary Keratinocyte Isolation and Culturing
		2.5 Reagents for Organoid Modeling
	3 Methods
		3.1 Human Dermal Papilla Isolation and Culturing
		3.2 Human Skin Keratinocyte Isolation and Culturing
		3.3 Organoid Modeling
			3.3.1 Cell Suspension Preparation
				Dermal Papilla Cell Suspension
				Keratinocyte Cell Suspension
				Primary Keratinocyte Medium
			3.3.2 Obtaining Organoids
	4 Notes
	References
Chapter 14: Method for Human Eccrine Sweat Gland Isolation from the Scalp by Means of the Micropunch Technique and Vital Dyes
	1 Introduction
		1.1 Anatomy and Distribution of Eccrine Glands in the Scalp
		1.2 Other Methods for Eccrine Gland Isolation
	2 Materials
		2.1 Local Anesthetic Infiltration
		2.2 Surgical Equipment for Follicular Unit Harvesting (See Note 1)
		2.3 Equipment for Eccrine Gland Microdissection
	3 Methods
		3.1 Anesthetic Infiltration
		3.2 Follicular Unit Harvesting
		3.3 Microscopic Dissection of the Eccrine Coil
	4 Notes
	References
Chapter 15: Identification of Long Noncoding RNA by In Situ Hybridization Approaches
	1 Introduction
	2 Materials
		2.1 In Situ Hybridization (ISH)
		2.2 Fluorescent In Situ Hybridization (FISH)
	3 Methods
		3.1 In Situ Hybridization (ISH)
			3.1.1 Tissue Preparation and Embedding
			3.1.2 Sectioning and Tissue Fixation
			3.1.3 Acetylation of the Tissue
			3.1.4 Hybridization and Slides Washing
			3.1.5 Antibody Incubation and Detection
		3.2 Fluorescent In Situ Hybridization (FISH)
			3.2.1 Fixation of Frozen Tissue
			3.2.2 Fixation of Adherent Cells
			3.2.3 Hybridization in Adherent Cells and/or Frozen Tissue
	4 Notes
	References
Chapter 16: Detection of MicroRNAs by In Situ Hybridization in Skin
	1 Introduction
	2 Materials
	3 Methods
		3.1 In Situ Hybridization on Frozen Tissue Sections
		3.2 In Situ Hybridization of Paraffin-Embedded Tissue Sections
	4 Notes
	References
Chapter 17: Chromatin Immunoprecipitation of Low Number of FACS-Purified Epidermal Cells
	1 Introduction
	2 Materials
		2.1 Skin Tissue Processing
		2.2 Viability Staining and Cell Fixation
		2.3 Purifying Epidermal Cells by FACS
		2.4 ChIP
		2.5 Stock Solutions
		2.6 Cell Lysis Buffers
		2.7 Wash and Elution Buffers
	3 Methods
		3.1 Dissection of Mouse Newborn (P0) Back Skin Samples, Preparation of Cell Suspension, and Prefixing
		3.2 Antibody Staining and FACS Sorting
		3.3 Nuclei Isolation
		3.4 Optimization of Chromatin Sonication
		3.5 Immunoprecipitation
		3.6 Washes and Elution
		3.7 DNA Purification and Quantification
		3.8 ChIP-qPCR and Assessment of Input Recovery Percentage
	4 Notes
	References
Chapter 18: 3D-FISH Analysis of the Spatial Genome Organization in Skin Cells in Situ
	1 Introduction
	2 Materials
		2.1 Amplification and Labeling of the DNA Probes by DOP-PCR
		2.2 DNA Amplification Utilizing Bacteriophage Phi29 DNA Polymerase
		2.3 Labeling the BAC or Phagemid DNA Probes by Nick Translation
		2.4 Tissue Fixation and Cryopreservation
		2.5 Preparation of the Tissue Sections for Hybridization
		2.6 Preparation of the DNA Probe Solution for Hybridization
		2.7 Hybridization Setup
		2.8 Posthybridization Tissue Processing
	3 Methods
		3.1 Labeling the DNA Probes
			3.1.1 Whole Chromosome Paint Amplification by DOP-PCR
			3.1.2 Whole Chromosome Paint Labeling by DOP-PCR
			3.1.3 BAC or Phasmid DNA Amplification Utilizing Bacteriophage Phi29 DNA Polymerase
			3.1.4 Labeling the BAC or Phagemid DNA Probes by Nick Translation
		3.2 Preparation of the Frozen Tissue Sections
			3.2.1 Tissue Fixation and Cryopreservation
			3.2.2 Preparation of the Tissue Sections for Hybridization
		3.3 DNA Hybridization
			3.3.1 Preparation of the DNA Probe Solution for Hybridization
			3.3.2 Hybridization Set  Up
		3.4 Post-Hybridization Tissue Processing
	4 Notes
	References
Chapter 19: Method to Study Skin Cancer: Two-Stage Chemically Induced Carcinogenesis in Mouse Skin
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 20: Investigation of Skin Wound Healing Using a Mouse Model
	1 Introduction
	2 Materials
	3 Methods
		3.1 Generation of Skin Wounds in Mice (See Note 1)
		3.2 Monitoring of the Wound Healing Process (See Note 5)
		3.3 Histological and Gene Expression Analysis
	4 Notes
	References
Chapter 21: Human Wound Healing Ex Vivo Model with Focus on Molecular Markers
	1 Introduction
	2 Materials
		2.1 Instruments and Tools
		2.2 William´s E Culture Medium
	3 Methods
		3.1 Ex Vivo Wounding
	4 Notes
	References
Chapter 22: Method for Investigation of Photobiological Effects of Light on Human Skin Cells Mediated by Low Doses of Light
	1 Introduction
	2 Materials
		2.1 Biomaterials
		2.2 Mechanical and Optical Components
		2.3 Metrology Equipment
	3 Methods
		3.1 Design of Light-Based Devices
			3.1.1 Optical Design
			3.1.2 Mechanical Design
			3.1.3 In-House Devices
		3.2 Quality Checks Before Executing an Experiment
			3.2.1 Check the Irradiance at the Position of the Cell Culture
			3.2.2 Check the Homogeneity of the Irradiation Pattern
			3.2.3 Check Light-Induced Thermal Impact on the Target
			3.2.4 Check a Potential Correlation Between Thermal Impact and Light Treatment
			3.2.5 Automated Control of the Light-Based Devices
			3.2.6 Check the Impact of the Surrounding Environment
			3.2.7 Have a Tight Control of the Biological Sample
		3.3 Design of Experiment
			3.3.1 Nomenclature
			3.3.2 Select Factors, Levels and Intervals for the Design of Experiment
		3.4 Robust Measurement of the Impact of Visible/NIR Light on Human Skin Cells
			3.4.1 Measurement Method Analysis
			3.4.2 Experimental Procedure
			3.4.3 Statistical Analysis and Visualization of the Data
	4 Notes
	References
Index