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Cardiac Stem Cell Niche, MMP9, and Culture and Differentiation of Embryonic Stem Cells

Embryonic stem cells (ESC) are totipotent, self-renewing, and clonogenic, having potential to differentiate into a wide variety of cell types. Due to regenerative capability, it has tremendous potential for treating myocardial infarction (death of myocardial tissue) and type 1 diab ...

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Unbiased Stereological Method to Assess Proliferation Throughout the Subependymal Zone

The subependymal zone (SEZ), frequently named as adult subventricular zone (SVZ), is a niche of adult neural stem and progenitor cells that lines a large extension of the lateral ventricles of the brain. The majority of the studies do not analyze the SEZ throughout its entire extension. Instead, st ...

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Looking for the Niche: Substance Delivery into the Lateral Ventricle of the Brain: The Osmotic Minipump System

The infusion of different substances into the left ventricle of the brain allows substances to reach the subventricular zone, one of the neural stem cell niches in the adult brain. Implantation of an osmotic minipump delivers proteins, virus and drugs directly into the lateral ventricle to act ...

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The Prospective Isolation of Viable, High Ploidy Megakaryocytes from Adult Murine Bone Marrow by Fluorescence Activated Cell Sorting

Mature megakaryocytes (MM) can be up to 65 μM in diameter and due to their size, viable and pure MM populations have been difficult to isolate in large numbers. Here in, we report a fluorescence activated cell sorting (FACS) method by which viable and pure populations of 8 N, 16 N, 32 N, and 64 N MM can be isolated from muri ...

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Microencapsulation of Stem Cells to Study Cellular Interactions

Microencapsulation is a technique used in both controlled delivery of materials over time as well as preservation of these materials while delivery is occurring. The range of materials able to be encapsulated is variable, from drugs to living cells. The latter is described here. Electrospr ...

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Proteomics Analysis of Contact-Initiated Eph ReceptorEphrin Signaling

Large-scale biochemical analysis of cell-specific signaling can be interrogated in cocultures of Eph receptor- and ephrin-expressing cells by combining proteomics analysis with cell-specific metabolic labeling. In this chapter, we describe how to perform such large-scale an ...

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Proteomic Analysis of the Left Ventricle Post-myocardial Infarction to Identify In Vivo Candidate Matrix Metalloproteinase Substrates

Left ventricular remodeling post-myocardial infarction (MI) involves a multitude of mechanisms that regulate the repair response. Matrix metalloproteinases (MMPs) are a major family of proteolytic enzymes that coordinate extracellular matrix turnover. MMP-7 or MMP-9 dele ...

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Generation and Analysis of Biosensors to Measure Mechanical Forces Within Cells

The inability to measure mechanical forces within cells has been limiting our understanding of how mechanical information is processed on the molecular level. In this chapter, we describe a method that allows the analysis of force propagation across distinct proteins within living cel ...

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Measuring CellCell Tugging Forces Using Bowtie-Patterned mPADs (Microarray Post Detectors)

Cells generate traction forces upon adhesion to the extracellular matrix as well as to neighboring cells. These forces are important for the growth and maintenance of adhesion structures such as focal adhesions and adherens junctions, and may play roles in tissue development. Here, we desc ...

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Micropatterning Cell Adhesion on Polyacrylamide Hydrogels

Cell shape and substrate rigidity play critical roles in regulating cell behaviors and fate. Controlling cell shape on elastic adhesive materials holds great promise for creating a physiologically relevant culture environment for basic and translational research and clinical ...

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Single Cell Analysis of Lipid Rafts

Lipid rafts are plasma membrane microdomains that serve as platforms for the assembly of proteins involved in signal transduction pathways. Given that lipid rafts are relatively resistant to cold extraction with nonionic detergents, lipid raft associated and nonassociated prot ...

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Cell-Surface ProteinProtein Interaction Analysis with Time-Resolved FRET and Snap-Tag Technologies

F�rster resonance energy transfer (FRET) is a proximity-dependent quantum effect that allows the measurement of protein interactions and conformational changes which are invisible to traditional forms of fluorescence or electron microscopy. However, FRET experiments oft ...

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Paracrine Communication Between Mechanically Stretched Myocytes and Fibroblasts

Mechanical stretch is a major factor for myocardial hypertrophy and heart failure. Stretch activates mechanical sensors from cardiac myocytes, leading to a series of signal transduction cascades, which can result in cell malfunction and remodeling. It is well known that mechanical st ...

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Production of Spontaneously Beating Neonatal Rat Heart Tissue for Calcium and Contractile Studies

Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FB) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Typically, these cell types are separated using Percoll density g ...

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Analyzing CellCell Interactions in 3-Dimensional Adhesion Assays

The organization of cells is key to the proper formation and function of tissues and it appears to be dependent upon various intracellular and extracellular signals. These signals come from cell–cell interactions, as well as interactions with the surrounding extracellular milieu. In or ...

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Control of Vascular Tube Morphogenesis and Maturation in 3D Extracellular Matrices by Endothelial Cells and Pericytes

An important advance using in vitro EC tube morphogenesis and maturation models has been the development of systems using serum-free defined media. Using this approach, the growth factors and cytokines which are actually necessary for these events can be determined. The first model devel ...

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Cell Membrane Vesicles as a Tool for the Study of Direct EpithelialStromal Interaction: Lessons from CD147

Communication between the epithelial and stromal tissue layers, separated by basement membrane, is known to provide the information necessary for development, differentiation, and homeostasis. These interactions are altered in benign or malignant diseases, in particular wh ...

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In Vivo Quantification of Metastatic Tumor Cell Adhesion in the Pulmonary Microvasculature

In vivo and ex vivo fluorescence video microscopy used to be a well-established method in life science with a variety of applications, such as in inflammation or cancer research. In this book chapter, we describe a model of in vivo fluorescence microscopy of the rat’s lung with the exclusive advanta ...

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Assessing BloodBrain Barrier Function Using In Vitro Assays

The impermeability of the blood–brain barrier (BBB) is due to a number of properties including tight junctions on adjoining endothelial cells, absence of pinocytic vesicles, and expression of multidrug transporters. Although the permeability of many chemicals can be predicted by th ...

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A High Content Imaging-Based Approach for Classifying Cellular Phenotypes

Current methods to characterize cell–biomaterial interactions are population-based and rely on imaging or biochemical analysis of end-point biological markers. The analysis of stem cells in cultures is further challenged by the heterogeneous nature and divergent fates of stem c ...

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