Springer protocols feed by Molecular Medicine 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Identification of microRNAs in Human Plasma
In recent years, microRNAs (miRNA) have been demonstrated to be present in body fluids and may therefore serve as diagnostic markers for diseases. By characterizing miRNA profiles in plasma, a miRNA signature may potentially be developed as a diagnostic and risk assessment tool for particular (patho)physiological states. This chapter describes the isolation, purification, identification, and sequencing of human plasma miRNAs. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Profiling Histone Modifications by Chromatin Immunoprecipitation Coupled to Deep Sequencing in Skeletal Cells
Chromatin, tightly packaged genomic DNA, is reliant on posttranslational modification of histone N-terminal tails for accessibility of DNA by transcription factors to activate transcription. Each histone modification may denote permissible states for gene activation or repression. As cells undergo differentiation, as they do in the skeleton from multipotential precursors through osteoblasts and into osteocytes, their histone code may be altered to help accommodate these transitions. Here we describe the methodology of chromatin immunoprecipitation (ChIP) coupled to deep sequencing (ChIP-seq) on skeletal cells that have dif...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Generation of Induced Pluripotent Stem Cells
Induced pluripotent stem cells (iPSCs) are generated from somatic cells that have been reprogrammed by the ectopic expression of defined embryonic transcription factors. This technology has provided investigators with a powerful tool for modelling disease and developing treatments for human disorders. This chapter provides the researcher with some background on iPSCs and details on how to produce MEF-conditioned medium, prepare mitotically arrested mouse embryonic fibroblasts (MEFs), create iPSCs using viral vectors, passage iPSCs, and cryopreserve iPSCs. The methods offered here have been used in many laboratories around ...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Engineering Cartilage Tissue by Pellet Coculture of Chondrocytes and Mesenchymal Stromal Cells
Coculture of chondrocytes and mesenchymal stromal cells (MSCs) in pellets has been shown to be beneficial in engineering cartilage tissue in vitro. In these cultures trophic effects of MSCs increase the proliferation and matrix deposition of chondrocytes. Thus, large cartilage constructs can be made with a relatively small number of chondrocytes. In this chapter, we describe the methods for making coculture pellets of MSCs and chondrocytes. We also provide detailed protocols for analyzing coculture pellets with cell tracking, proliferation assays, species specific polymerase chain reactions (PCR), short tandem repeats anal...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Isolating Endosteal Mesenchymal Progenitors from Rodent Long Bones
Bone marrow mesenchymal stem cells (MSCs) are promising therapeutic tools for tissue repair and the treatment of a number of human diseases. As a result, there is substantial interest in characterizing and expanding these cells to uncover their therapeutic potential. For preclinical studies, mesenchymal progenitors, containing both MSCs and their proliferative progeny, are commonly isolated from the central region of rodent long bones. However, challenges exist in expanding these central mesenchymal progenitors in culture. We have recently identified another population of progenitors within rodent long bones that resides c...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Primary Murine Growth Plate and Articular Chondrocyte Isolation and Cell Culture
The ability to isolate primary chondrocytes from wild-type and genetically altered mice has provided tremendous advances in the understanding of signaling networks that regulate chondrocytes in health and disease. Isolation of chondrocytes from both growth plate tissues and articular cartilage has been challenging due to the cells being embedded within a highly organized tissue matrix. Here we describe highly reproducible methods for the isolation of pure populations of growth plate chondrocytes from the murine sternum and ribs and articular chondrocytes from the knee joint. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Isolation of Osteocytes from Mature and Aged Murine Bone
Osteocytes are thought to be the mechanosensors of bone by sensing mechanical loads imposed upon the bone and transmitting these signals to the other bone cells to initiate bone modeling and remodeling. The location of osteocytes deep within bone is ideal for their function. However, this location makes the study of osteocytes in vivo technically difficult. There are several methods for obtaining and culturing primary osteocytes for in vitro experiments and ex vivo observation. In this chapter, several proven methods are discussed including the isolation of avian osteocytes from chicks and osteocytes from calvaria and long...
Source: Springer protocols feed by Molecular Medicine - October 24, 2014 Category: Molecular Biology Source Type: news
Basic Techniques for Studies of iNKT Cells and MAIT Cells
Invariant natural killer T (iNKT) cells and mucosal-associated invariant T cells (MAIT cells) are T cell subsets belonging to innate-like lymphocytes. These innate-like lymphocytes express semi-invariant T cell receptors, but exert diverse functions and thus are involved in various types of immune responses. As iNKT cells and MAIT cells are abundant in human peripheral blood, these cells may hold important physiological roles, and thus it is desired to reveal their functions. Here, we first describe the cell preparation techniques commonly used in studies of innate-like lymphocytes, and then introduce methods for the detec...
Source: Springer protocols feed by Molecular Medicine - April 7, 2014 Category: Molecular Biology Source Type: news
Studies on the T Cell Receptor (TCR) Revision of Autoantibody-Inducing CD4 T (aiCD4 T) Cell
Our recent studies into the role of autoantibody-inducing CD4 T cells in autoimmune disease have necessitated studies on the mechanism of TCR revision, a phenomenon that has been difficult to approach experimentally. Here we describe a detailed experimental technique to investigate the molecular events involved in TCR revision. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - April 7, 2014 Category: Molecular Biology Source Type: news
Characterization of MicroRNAs and Their Targets
MicroRNAs (miRNAs) have emerged as key players in the degradation of target mRNAs. They have been associated with diverse biological processes, and recent studies have demonstrated that miRNAs play a role in inflammatory responses. The identification of miRNA and their corresponding messenger RNA (mRNA) targets can therefore be very helpful. In this chapter, we first overview the field of miRNAs and then show the fundamental techniques for the identification of miRNAs and confirmation of their role on target mRNAs. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - April 7, 2014 Category: Molecular Biology Source Type: news
In Vivo Cell Transfer Assay to Detect Autoreactive T Cell Subsets
Among the methods used in molecular biology, in vitro biochemical assays are more common, whereas in vivo assays, including the use of animal models, are less widely employed. In our studies on systemic lupus erythematosus (SLE), we have identified a novel T cell subtype termed “autoantibody-inducing CD4 T cells” (aiCD4 T cell) that is responsible for the development of autoimmunity. In order to identify and isolate these cells, we developed a new technique that involves the transfer of candidate T cell subpopulations into naïve mice and assaying for the development of autoantibodies in the recipient mice....
Source: Springer protocols feed by Molecular Medicine - April 7, 2014 Category: Molecular Biology Source Type: news
Unraveling Autoimmunity with the Adoptive Transfer of T Cells from TCR-Transgenic Mice
Transgenesis of rearranged α and β chains from the T-cell receptor has allowed the generation of a variety of mice with a predetermined T-cell repertoire. These mice have been extensively used as tools to circumvent the low precursor frequency of naturally occurring endogenous T cells. As such, they have been valuable to study pathways of T-cell development in the thymus. In addition, these mice can also be considered as a valuable source of naive and/or memory T cells with a defined specificity. I will comment in this chapter the use of this source of T cells with known antigen reactivity to study in vivo T-cel...
Source: Springer protocols feed by Molecular Medicine - April 7, 2014 Category: Molecular Biology Source Type: news
Principles for the Use of In Vivo Transgene Techniques: Overview and an Introductory Practical Guide for the Selection of Tetracycline-Controlled Transgenic Mice
Transgenic mice are a beneficial tool that can allow researchers to investigate the roles of specific genes in physiology and disease. However, conventional transgenic mice have the limitation that constitutive expression of a transgene from the embryonic stage may affect the normal development of the mice or cause compensating effects. To overcome these disadvantages, tetracycline-controlled transgenic mice, which can express target gene products in a tissue-specific and time-dependent manner, have been developed. In this section, the principles of tetracycline-controlled systems are discussed first. In addition, useful i...
Source: Springer protocols feed by Molecular Medicine - April 7, 2014 Category: Molecular Biology Source Type: news
Characterization of Innate Immune Signalings Stimulated by Ligands for Pattern Recognition Receptors
The innate immunity is an essential step as the front line of host defense, and its aberrant activation particularly in response to nucleic acids is closely related to the pathogenesis of autoimmune and inflammatory diseases. Characterization of the innate immune signalings may provide a pathophysiological insight for better understanding of human diseases. Nucleic acid-mediated activation of pattern recognition receptors triggers the activation of two major intracellular signaling pathways, which are dependent on NF-κB and interferon regulatory factors, transcriptional factors. This leads to the subsequent induction...
Source: Springer protocols feed by Molecular Medicine - April 7, 2014 Category: Molecular Biology Source Type: news
Monitoring Multifunctionality of Immune-Exhausted CD8 T Cells in Cancer Patients
CD8 T cells play a critical role in the host defense against cancers and infectious diseases. However, the presence of antigen-specific CD8 T cells does not always imply that cancers and/or pathogens are efficiently eliminated in the body. Concerning this point, the recent studies suggest the concept of immune exhaustion of CD8 T cells, characterized by their decreased production of IL-2, TNFα, and IFNγ even after antigen stimulation. Thus, continuous stimulation of CD8 T cells by the persistent antigens results in immune exhaustion, which eventually causes immune tolerance against cancers and chronic infection...
Source: Springer protocols feed by Molecular Medicine - April 7, 2014 Category: Molecular Biology Source Type: news
