Methods in molecular biology (Clifton, N.J.) (Meth Mol Biol )

Publisher: Humana Press

Description

  • Impact factor
    1.29
  • 5-year impact
    0.00
  • Cited half-life
    0.00
  • Immediacy index
    0.00
  • Eigenfactor
    0.00
  • Article influence
    0.00
  • Other titles
    Methods in molecular biology (Clifton, N.J.), Methods in molecular biology
  • ISSN
    1940-6029
  • OCLC
    24839341
  • Material type
    Series
  • Document type
    Journal / Magazine / Newspaper

Publisher details

Humana Press

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  • Post-print
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    • On funders designated website/repository after 12 months
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    • Set phrase to accompany link to published version (The original publication is available at www.springerlink.com)
    • Articles in some journals can be made Open Access on payment of additional charge
    • 'Humana Press (Springer Imprint)' is an imprint of 'Springer Verlag (Germany)'
  • Classification
    ​ green

Publications in this journal

  • [Show abstract] [Hide abstract]
    ABSTRACT: Notch signaling is probably the most widely used intercellular communication pathway. The Notch mutant in the fruit fly Drosophila melanogaster was isolated about 100 years ago at the dawn of genetics. Since then, research on Notch and its related genes in flies, worms, mice, and human has led to the establishment of an evolutionarily conserved signaling pathway, the Notch signaling pathway. In the past few decades, molecular cloning of the Notch signaling components as well as genetic, cell biological, biochemical, structural, and bioinformatic approaches have uncovered the basic molecular logic of the pathway. In addition, genetic screens and systems approaches have led to the expansion of the list of genes that interact and fine-tune the pathway in a context specific manner. Furthermore, recent human genetic and genomic studies have led to the discovery that Notch plays a role in numerous diseases such as congenital disorders, stroke, and especially cancer. Pharmacological studies are actively pursuing key components of the pathway as drug targets for potential therapy. In this chapter, we will provide a brief historical overview of Notch signaling research and discuss the basic principles of Notch signaling, focusing on the unique features of this pathway when compared to other signaling pathways. Further studies to understand and manipulate Notch signaling in vivo in model organisms and in clinical settings will require a combination of a number of different approaches that are discussed throughout this book.
    Methods in molecular biology (Clifton, N.J.) 09/2014; 1187:1-14.
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    ABSTRACT: An essential parameter to evaluate the light emission properties of fluorophores is the fluorescence quantum yield, which quantify the conversion efficiency of absorbed photons to emitted photons. We detail here an alternative nonfluorescent method to determine the absolute fluorescence quantum yield of quantum dots (QDs). The method is based in the so-called Thermal Lens Spectroscopy (TLS) technique, which consists on the evaluation of refractive index gradient thermally induced in the fluorescent material by the absorption of light. Aqueous dispersion carboxyl-coated cadmium telluride (CdTe) QDs samples were used to demonstrate the Thermal Lens Spectroscopy technical procedure.
    Methods in molecular biology (Clifton, N.J.) 08/2014; 1199:93-101.
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    ABSTRACT: As well in light as in transmission electron microscopy can be seen that the renal stem/progenitor cell niche shows a special arrangement of two different kinds of stem/progenitor cells. Epithelial cells are found in the tip of an ureteric bud derived CD ampulla encircled by a special basal lamina. Mesenchymal cells are separated from them by a striking interstitial interface. Specimens fixed by conventional glutaraldehyde solution show that the interface looks bright and unremarkable. In contrast, fixation of specimens with glutaraldehyde in combination with cupromeronic blue, ruthenium red, or tannic acid illustrates that the interface contains a remarkable network of extracellular matrix spanning between epithelial and mesenchymal stem/progenitor cells. After unpacking this particular extracellular matrix for electron microscopy, elaboration of related functions such as structural composition of contained molecules, binding of morphogenetic factors, and influence on parenchyma development is under current experimental work.
    Methods in molecular biology (Clifton, N.J.) 07/2014;
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    ABSTRACT: The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential.Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged.Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems.
    Methods in molecular biology (Clifton, N.J.) 07/2014;
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    ABSTRACT: Immunoassays represent valuable and broadly used techniques for detection and quantification of proteins. Thanks to their high sensitivity, such techniques are powerful for analyzing growth factors, trophic factors, angiogenic factors, hormones, cytokines, chemokines, soluble receptors, and other proteins which play key roles in intercellular communication and operate as potent regulators of stem cell survival, proliferation, differentiation, or cell death. Multiplex immunological assays, in contrast to ELISA, offer simultaneous quantification of tens of proteins across multiple samples, and have been developed to save time, costs, and sample volumes. Among them, planar antibody microarrays and xMAP(®) bead-based assays have become particularly popular for characterization of proteins secreted by stem cells, as they are relatively easy, highly accurate, multiplex to a high degree and a broad spectrum of analytes can be measured. Here, we describe protocols for multiplex quantification of secreted proteins using Quantibody(®) microarrays (RayBiotech) and xMAP(®) assays (Luminex and its partners).
    Methods in molecular biology (Clifton, N.J.) 07/2014;
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    ABSTRACT: Stem cells are unspecialized cells that can self-renew and have the ability to develop into cells of highly specialized functions. The study of stem cells holds enormous promise in the medical field ranging from their uses in cell therapies to their uses for greater understanding of tissue development and disease pathologies. Stem cells have been isolated from tendon tissue recently. These tendon-derived stem cells (TDSCs) are particularly relevant for tendon repair and the study of the potential roles of stem cells in tendon pathology as they are isolated from tendon tissues. This paper aims to describe the step-by-step protocol and the practical tips for the isolation and verification of stem cell characteristics of TDSCs. The cell seeding density and hence cell-cell contact has a significant impact on the isolation and expansion of TDSCs. Hence, I also describe our established protocol for the determination of the optimal seeding density for TDSC isolation and culture.
    Methods in molecular biology (Clifton, N.J.) 07/2014;
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    ABSTRACT: Experimental autoimmune encephalitis (EAE), the animal model of multiple sclerosis (MS), has provided significant insight into the mechanisms that initiate and drive autoimmunity. Several central nervous system proteins and peptides have been used to induce disease, in a number of different mouse strains, to model the diverse clinical presentations of MS. In this chapter, we detail the materials and methods used to induce active and adoptive EAE. We focus on disease induction in the SJL/J, C57BL/6, and BALB/c mouse strains, using peptides derived from proteolipid protein, myelin basic protein, and myelin oligodendrocyte glycoprotein. We also include a protocol for the isolation of leukocytes from the spinal cord and brain for flow cytometric analysis.
    Methods in molecular biology (Clifton, N.J.) 07/2014;
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    ABSTRACT: Multiple sclerosis (MS) is the most common inflammatory, demyelinating, and neurodegenerative disorder of the central nervous system (CNS) in humans. Although the etiology of MS remains unknown, several lines of evidence support the notion that autoimmunity against components of the myelin sheath plays a major role in susceptibility to and development of the disease. At present, there are no approved MS therapies aimed specifically toward downregulating antigen-specific autoreactive immune cells. One antigen-specific approach that appears promising for the treatment of MS is DNA vaccination. This technique has demonstrated efficacy in clinical trials while maintaining safety.Here, we describe the generation of DNA vaccines containing immunologically relevant antigens of MS. Moreover, we present a detailed protocol for the prophylactic and therapeutic administration of DNA vaccines via intramuscular injection targeting on the development of experimental autoimmune encephalomyelitis (EAE), an animal model resembling MS.
    Methods in molecular biology (Clifton, N.J.) 06/2014;
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    ABSTRACT: Human embryonic stem cells are pluripotent cells typically derived from blastulating embryos that have become excess to clinical needs in assisted reproduction programs. They provide cellular models for embryonic development and disease, and are thought to be useful for future cell replacement therapies and regenerative medicine. Here we describe methods to derive human embryonic stem cell lines. This includes blastocyst cryopreservation using a highly efficient vitrification protocol, the production and use of fibroblast feeder cells, embryo plating and passaging of resulting cellular outgrowths, and cryopreservation of putative stem cells lines.
    Methods in molecular biology (Clifton, N.J.) 06/2014;
  • Methods in molecular biology (Clifton, N.J.) 06/2014; 1171:47.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human-induced pluripotent stem cells, are a renewable cell source for a wide range of applications in regenerative medicine and useful tools for human disease modeling and drug discovery. For these purposes, large numbers of high-quality cells are essential. Recently, we showed that a biological substrate, recombinant E8 fragments of laminin isoforms, sustains long-term self-renewal of hPSCs in defined, xeno-free medium with dissociated single-cell passaging. Here, we describe a modified culture system with similar performance to efficiently expand hPSCs under defined, xeno-free conditions using a non-biological synthetic substrate.
    Methods in molecular biology (Clifton, N.J.) 05/2014;
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    ABSTRACT: In vitro proliferation of hematopoietic stem cells (HSCs) is yet an unresolved challenge. Found in the bone marrow, HSCs can undergo self-renewing cell division and thereby multiply. Recapitulation of the bone marrow environment in order to provide the required signals for their expansion is a promising approach.Here, we describe a technique to produce biofunctionalized, macroporous poly(ethylene glycol) diacrylate (PEGDA) hydrogels that mimic the spongy 3D architecture of trabecular bones, which host the red, blood-forming bone marrow. After seeding these scaffolds with cells, they can be used as simplified bone marrow analogs for the cultivation of HSCs. This method can easily be conducted with standard laboratory chemicals and equipment. The 3D hydrogels are produced via salt leaching and biofunctionalization of the material is achieved by co-polymerizing the PEGDA with an RGD peptide. Finally, cell seeding and retrieval are described.
    Methods in molecular biology (Clifton, N.J.) 05/2014;
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    ABSTRACT: Stem cell-based therapies have drawn intensive attention in the neuronal regenerative fields. Several studies have revealed that stem cells can serve as an inexhaustible source for neurons for transplantation therapies. However, generation of neurons and directionality has not yet been fully investigated. Herein, we investigate the mechanical ramifications of surface topography on human embryonic cell differentiation. Microgrooved surfaces with various pitches were applied to modulate the neuron differentiation. Our protocol showed that neuron differentiation increased as grove pitch decreased. The results indicated that 2 μm microgrooves can improve neuron growth by ~1.7-fold. Our results indicate the importance of mechanotransduction on neuronal differentiation and highlight the feasibility of manipulating the neuronal differentiation with surface topography, providing new perspectives for accommodating clinical transplantation.
    Methods in molecular biology (Clifton, N.J.) 05/2014;
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    ABSTRACT: In this chapter information is provided about the outer layer of the skin, the epidermis, and the predominant cells comprising this epithelium, the keratinocytes. The evidence supporting a possible role for the lipid-metabolizing enzyme phospholipase D in regulating keratinocyte differentiation is also discussed. A detailed protocol for the preparation of primary cultures of epidermal keratinocytes from neonatal mice is described, to allow other investigators to obtain data concerning these important cells involved in forming and maintaining the mechanical and water permeability of the skin. Finally, a complete protocol for monitoring phospholipase D activity in intact cells is supplied in the hope that additional research will result in a better understanding of the role of phospholipase D in controlling keratinocyte proliferation and differentiation.
    Methods in molecular biology (Clifton, N.J.) 05/2014;
  • Methods in molecular biology (Clifton, N.J.) 04/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: In many tissues, cells must be aligned for proper function. This alignment can occur at the cellular and/or subcellular (protein/molecular) level. The alignment of cytoskeletal components, in fact, precedes whole cell alignment. A variety of methods exist to manipulate cytoskeletal and whole cell alignment; one of the simplest and most predictable involves seeding adherent cells onto defined substrate topography. We present here two methods to create grooved multiwell plates: one involving microfabrication, which allows for custom design of substrate topography, and a simpler, inexpensive method using commercially available diffraction gratings. We also include methods for manual and automatic quantification of cell alignment.
    Methods in molecular biology (Clifton, N.J.) 04/2014;
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    ABSTRACT: Cell aggregate culture is a widely used, reliable system for promoting chondrogenic differentiation of stem cells. A high-throughput cell pellet culture enables screening of various soluble factors for their effects on stem cell function and chondrogenesis. In this protocol, we report a platform that allows the formation of stem cell aggregates in a 96-well plate format. Specifically, stem cells are centrifuged to form high-density pellets, mimicking mesenchymal condensation. The cell aggregates can be differentiated into chondrocytes when cultured in chondrogenic medium for 4 weeks. Such a technique is compatible for high-throughput screening and can be very useful for optimizing conditions for cartilage tissue engineering.
    Methods in molecular biology (Clifton, N.J.) 03/2014;
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    ABSTRACT: Eukaryotic DNA is wrapped around histone octamers, known as nucleosomes, in an orderly fashion that provides the primary structure of chromatin organization. The compaction of DNA into nucleosomal repeats not only allows the tight packaging of the large eukaryotic genomes into the nucleus, it also dictates the accessibility of genetic information. Thus, in order to understand how nucleosomes can affect the dynamics of DNA-protein interactions, such as those associated with transcriptional regulatory mechanisms, it is important to define nucleosomal positioning and occupancy along genomic DNA. Here we describe a method that relies on the enzymatic activity of micrococcal nuclease (MNase) to determine nucleosomal footprints and boundaries. By pairing this technique with next generation sequencing techniques (i.e., MNase-seq), it is possible to generate a genome-wide detailed map of chromatin architecture.
    Methods in molecular biology (Clifton, N.J.) 03/2014;

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