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Analysis of Cell Movement Between Skin and Other Anatomical Sites In Vivo Using Photoconvertible Fluorescent Protein Kaede-Transgenic Mice

Clarification of the spatiotemporal regulation and function of immune cells within the skin is critical to the understanding of the role of immune cells and the skin in immune homeostasis. Here, we describe a novel assay system for monitoring cell movements in the entire body using the photocon ...

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Detection, Enumeration, and Characterization of Immune Cells Infiltrating Melanoma Tumors

Tumor-infiltrating immune cells have long been thought to affect tumor growth. In recent years, large retrospective studies have shown that the nature and polarization of the immune cells found within the tumor microenvironment impact not only the growth of the primary tumor, but also dise ...

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Isolation of Melanoma Tumor-Initiating Cells from Surgical Tissues

A new model of cancer progression has been put forward that predicts existence of tumor stem cells (TSCs) in the heterogeneous bulk tumor mass that self-renew, are resistant to chemo- and radiotherapies, and sustain tumor growth during the course of its progression or relapse (Ailles and Weiss ...

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Analysis of Collective Invasion of Carcinoma Cells in a 3D Organotypic Model

Cancer cell invasion and dissemination from primary tumors are complex multistep mechanisms which remain poorly understood. It is now clear that cancer cells can adapt their mode of invasion to the signalling provided by the surrounding stroma. Single and collective cancer cell invasi ...

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Induction of Granulocyte-Dependent Dermal-Epidermal Separation by Autoantibodies Ex Vivo

Disease models represent powerful tools used by biomedical researchers to address various questions related to the pathogenesis and the possible therapeutic targets in different diseases. To get a complete picture of the process one needs complementary animal and ex vivo disease mo ...

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In Vitro Pathogenicity Assay for Anti-desmoglein Autoantibodies in Pemphigus

Patients with pemphigus have circulating anti-desmoglein (Dsg) 1 and/or Dsg3 autoantibodies that induce blister formation on the skin and mucous membrane. We describe here two assays that measure the pathogenic strength of autoantibodies in blister formation: an in vitro dissocia ...

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Cell Death in the Skin: How to Study Its Quality and Quantity

The characterization of the quality and quantity of cell death has gained substantial interest over the past decades. More recently necroptosis as a programmed form of necrosis has been identified as an important additional form of cell death with relevance in the skin. Understanding how to a ...

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MicroRNA Profiling During Human Keratinocyte Differentiation Using a Quantitative Real-Time PCR Method

The terminal differentiation of epidermal keratinocytes requires transcriptional and posttranscriptional regulatory mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that play key roles during differentiation processes by regulating protein expression at ...

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Global Proteome Analyses of SILAC-Labeled Skin Cells

Physiological functions of skin cells are often altered in diseases. Since the underlying molecular mechanisms are generally executed by proteins, it is of interest to assess protein dynamics in normal and pathologically altered cells. These can be readily analyzed in relevant cell cu ...

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Dissecting and Culturing and Imaging the Mouse Urogenital System

Current knowledge of the morphological and molecular events driving branching morphogenesis of the ureteric bud (UB) during development of the metanephric kidney has been greatly facilitated by the ability to explant this organ to culture. The UB can be further isolated from the mesench ...

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In Vitro Culture of Embryonic Kidney Rudiments and Isolated Ureteric Buds

In vitro culture of embryonic kidney rudiments has been utilized to study a variety of cellular processes and developmental mechanisms. Here, we describe two-dimensional (2D) culture of embryonic kidney rudiments on Transwell filters and three-dimensional (3D) cultures in collag ...

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Live Imaging of the Developing Mouse Mesonephros

Embryonic development is a highly dynamic process involving complex tissue interactions and movements. Recent progress in cell labeling, image acquisition, and image processing technologies has brought the study of embryo morphogenesis to another level. It is now possible to visu ...

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In Vitro Induction of Nephrogenesis in Mouse Metanephric Mesenchyme with Lithium Introduction and Ureteric Bud Recombination

The organ culture setup of embryonic kidney has served as a model of nephrogenesis for several decades. In vitro culture of the mouse metanephric mesenchyme enables easy manipulation and analysis of the tissue and provides information of cellular interactions, morphogenesis, cell di ...

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Use of In Situ Hybridization to Examine Gene Expression in the Embryonic, Neonatal, and Adult Urogenital System

Studies into the molecular basis of morphogenesis frequently begin with investigations into gene expression across time and cell type in that organ. One of the most anatomically informative approaches to such studies is the use of in situ hybridization, either of intact or histologically ...

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Laser Capture

This chapter describes detailed methods used for laser capture microdissection (LCM) of discrete subpopulations of cells. Topics covered include preparing tissue blocks, cryostat sectioning, processing slides, performing the LCM, and purification of RNA from LCM samples. Not ...

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Isolation of High Quality RNA from Embryonic Kidney and Cells

All the mRNAs within a cell and their relative levels are indicative of gene expression within that cell, which is essential for its structure and function in its differentiated state. Therefore, methods for the identification of the specific mRNAs and the quantitation of their levels are inv ...

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Access and Use of the GUDMAP Database of Genitourinary Development

The Genitourinary Development Molecular Atlas Project (GUDMAP) aims to document gene expression across time and space in the developing urogenital system of the mouse, and to provide access to a variety of relevant practical and educational resources. Data come from microarray gene ex ...

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Engineered Tissues to Quantify Collective Cell Migration During Morphogenesis

Renal development is a complex process involving the dynamic interplay of over 25 different cell types. One distinct step in this process is the formation of the ureteric tree, which develops from the repeated branching of the ureteric bud. During branching of the ureteric bud, cells migrate col ...

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Making Immortalized Cell Lines from Embryonic Mouse Kidney

Immortalized cell lines derived from embryonic mouse kidneys are useful tools for exploring signaling pathways, morphogenetic mechanisms, and gene function in renal development: they also provide a means for efficient first-round screening of panels of small molecules intended ...

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Investigating Primary Cilia in Cultured Metanephric Mesenchymal Cells

Primary cilia are present in most vertebrate cells. They have complex structures that are required for signal transduction in developing tissues. The embryonic kidney consists of two major cell lineages, ureteric and metanephric mesenchyme. Here, we describe a method to isolate metan ...

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