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Preparation of Adult Muscle Fiber-Associated Stem/Precursor Cells

In our studies of muscle regeneration we have developed, modified, and optimized techniques to isolate and study the stem and precursor cells to muscle tissue. Our goals have been to obtain for study muscle fibers in bulk, or the fiber-associated cells, separately from the other cells found in musc ...

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Altered Expression of NFkB in Ex Vivo Differentiated Dendritic Cells from the Aged Subjects: Implications in Immunotherapy

Dendritic cells are the key initiators and organizers of the immune response. They are specialized antigen-presenting cells whose immunogenicity leads to the induction of antigen-specific immune responses. They are therefore ideal vehicles to deliver strong immunogenic sign ...

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Membrane Protein Expression in Cell-Free Systems

Cell-free expression has emerged as a promising tool for the fast and efficient production of membrane proteins. The rapidly growing number of successfully produced targets in combination with the continuous development of new applications significantly promotes the distribu ...

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Expression of Mammalian Membrane Proteins in Mammalian Cells Using Semliki Forest Virus Vectors

One of the major bottlenecks in drug screening and structural biology on membrane proteins has for a long time been the expression of recombinant protein in sufficient quality and quantity. The expression has been evaluated in all existing expression systems, from cell-free translation ...

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Preparation and Analysis of Endothelial Progenitor Cells Associated with Angiogenesis

Endothelial progenitor cell-based therapy offers great potential to reduce morbidity and mortality in patients with vascular diseases. In animal models of ischemia, circulating endothelial progenitor cells (EPCs) have been shown to home to sites of active angiogenesis and diffe ...

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Culture of Bone Marrow Mesenchymal Stem Cells on Engineered Matrix

Mesenchymal stem cells (MSCs) have huge potential to be used in cell therapies because of their pluripotency and immunoregulatory attributes. To harness and maximize the therapeutic potential of MSCs, a thorough understanding of their differentiation pathways and their respons ...

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Viral Packaging and Transduction of Adult Hippocampal Neural Progenitors

Genetic manipulation of adult hippocampal neural progenitor cells is a useful technique for exploring gene function through gain of function and loss of function mutations or RNAi. Furthermore, the introduction of new genes can “re-program” progenitor cell behavior to force a desired l ...

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In Vivo Analysis of Engrafted Adult Hippocampal Neural Progenitors

A neural degenerative disease is characterized by the deterioration of neural tissue and subsequent loss of function. The in vivo engraftment of neural stem cells is a promising approach to the functional replacement of neural tissue with the ultimate goal of regaining lost function. In add ...

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In Vitro Culture and Analysis of Adult Hippocampal Neural Progenitors

Adult hippocampal neural progenitor cell (AHNPC) culture is a useful technique for gaining insight into adult neurogenesis, studying disease, and high throughput drug screening. The ability of AHNPCs to proliferate and differentiate into the three cell lineages of the adult brain in ce ...

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Isolation of Adult Hippocampal Neural Progenitors

Adult neurogenesis, or the creation of new neurons in adult organisms, is an exciting recent area of neurological research. The subgranular zone of the adult hippocampus is one area where hippocampal neural progenitors generate new neurons that functionally integrate into existing n ...

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Use of Stem Cell Markers in Dissociated Mammary Populations

The regenerative potential of mammary epithelium facilitates assessment of the “stemness” of any epithelial subpopulation in transplantation assays. Thus, mammary tissue can be dissociated into single cells, stained for cell surface markers of interest and classified using fl ...

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Limiting-Dilution Transplantation Assays in Mammary Stem Cell Studies

Mammary reconstitution assays can be used to measure the stem cell frequency within an epithelial population by transplanting increasingly diluted single-cell preparations of the population of interest. There are fundamental steps in the single-cell isolation protocol which ...

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In Situ Analysis of Cell Populations: Long-Term Label-Retaining Cells

The mammary gland consists of an epithelial ductal tree embedded in a fat pad. Adult mammary epithelium has been demonstrated to have outstanding regenerative potential, consistent with the presence of resident, adult stem cells. However, there are currently no bona fide markers to ident ...

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Preparation of Collagen-Coated Gels that Maximize In Vitro Myogenesis of Stem Cells by Matching the Lateral Elasticity of In Vivo Muscle

The physical nature of a cell’s microenvironment – including the elasticity of the surrounding tissue – appears to exert a significant influence on cell morphology, cytoskeleton, and gene expression. We have previously shown that committed muscle cells will develop sarcomeric stria ...

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Practical Considerations of Membrane Protein Instability during Purification and Crystallisation

Crystallisation of integral membranes requires milligrams of purified protein in a homogeneous, monodisperse state, and crucially, the membrane protein must also be fully functional and stable. The stability of membrane proteins in solution is dependent on the type of detergents us ...

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Membrane Protein Solubilization

A critical step in any in vitro analysis of membrane proteins is the solubilization of the membrane to extract the protein of interest in an active form to obtain an aqueous solution containing the membrane protein complexed with detergents and lipids in a form suitable for purification and furt ...

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Heterologous Expression and Affinity Purification of Eukaryotic Membrane Proteins in View of Functional and Structural Studies: The Example of the Sar

Heterologous SERCA1a Ca2+-ATPase (sarco-endoplasmic reticulum Ca2+-adenosine triphosphatase isoform 1a) from rabbit was expressed in yeast Saccharomyces cerevisiae as a fusion protein, with a biotin acceptor domain (BAD) linked to the SERCA C-terminus by a thrombin cleavage s ...

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Amphipols and Fluorinated Surfactants: Two Alternatives to Detergents for Studying Membrane Proteins In vitro

Handling integral membrane proteins in aqueous solutions traditionally relies on the use of detergents, which are surfactants capable of dispersing the components of biological membranes into mixed micelles. The dissociating character of detergents, however, most often caus ...

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Interaction of Lipids and Ligands with Nicotinic Acetylcholine Receptor Vesicles Assessed by Electron Paramagnetic Resonance Spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy is a powerful technique that permits the study of membrane-embedded proteins in its lipid environment by assessing the interaction of spin labels with the protein in its natural environment (i.e., native membranes) or in reconstit ...

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Utilization of Liposomes for Studying Drug Transfer and Uptake

On entry into the body of the patient, drugs have to overcome many barriers in order to reach the target. The knowledge of the ability of drugs to cross these barriers, which mostly consist of lipid membranes, is of utmost interest in pharmacy. High values of lipophilicity of a drug might be a good pre-requisi ...

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