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.In Vivo Visualization of (Auto)Immune Processes in the Central Nervous System of Rodents
The CNS is effectively shielded from the periphery by the blood–brain barrier (BBB) which limits the entry of cells and solutes. However, in autoimmune disorders such as multiple sclerosis, immune cells can overcome this barrier and induce the formation of CNS inflammatory lesions. Recently, two-photon laser scanning microscopy (TPLSM) has made it possible to visualize autoimmune processes in the living CNS in real time. However, along with a high microscopy standard, this technique requires an advanced surgical procedure to access the region of interest. Here, we describe in detail the necessary methodological steps...
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
Myelinating Cultures: An In Vitro Tool to Identify Demyelinating and Axopathic Autoantibodies
Myelinating cultures derived from embryonic spinal cord provide an invaluable tool to detect demyelinating and axopathic autoantibodies in clinical samples. A single preparation will provide a minimum of 200 individual cultures allowing 60 or more samples to be assayed in triplicate. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
Isolation of Central Nervous System (CNS) Infiltrating Cells
Leukocyte infiltration of the central nervous system (CNS) occurs under certain pathogenic conditions and most often results in severe disorders. Therefore, the isolation and analysis of such infiltrating cell populations is necessary for elucidating the underlying pathogenic mechanisms. Here we describe a simple and straightforward protocol for cell isolation from the inflamed CNS, which combines mechanical dissociation and enzymatic degradation of the tissue. Additionally, purification by Percoll gradient centrifugation provides a great yield of the infiltrating material. The isolated cells can be further used for downst...
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
Experimental Autoimmune Encephalomyelitis in Marmosets
Experimental autoimmune encephalomyelitis (EAE) in the common marmoset, a small-bodied Neotropical primate, is a well-known and validated animal model for multiple sclerosis (MS). This model can be used for exploratory research, i.e., investigating the pathogenic mechanisms involved in MS, and applied research, testing the efficacy of new potential drugs. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
ELISPOT Techniques
The enzyme-linked immunospot (ELISPOT) assay is a widely used method for enumerating antigen-specific cytokine-producing or antibody-secreting immune cells. It is one of the most effective immunological and diagnostic approaches to detect and quantify low-frequency cytokine- or antibody-producing cells in human and animal tissues, such as peripheral blood, lymph nodes, and spleen. Detection and quantification of specific cytokine-producing cells by the ELISPOT assay is based on the formation of visible spots at the site of cytokine release by the cells under investigation (e.g., T cells) using pairs of different capture an...
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
Neuropathological Techniques to Investigate CNS Pathology in Experimental Autoimmune Encephalomyelitis (EAE)
Neuropathological techniques such as conventional and immunohistochemical staining of paraffin-embedded tissue sections are instrumental for identification and characterization of aberrations of organ architecture during human inflammatory disorders of the central nervous system (CNS) as in their animal models. Here we describe step-by-step protocols for tissue processing, sectioning, and conventional and immunohistochemical stainings to display as well as quantify CNS inflammation, demyelination, and neuronal damage in experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple Sclerosis (MS). (Source: Sp...
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
Positional Gene Cloning in Experimental Populations
We describe experimental populations and designs that can be used for positional cloning, including backcrosses, intercrosses, and heterogeneous stocks, and advantages and disadvantages of different approaches. Once the phenotype and genotype of each individual in an experimental population have been determined, QTL identification can be accomplished. We describe the statistical tools used to identify the existence, location, and significance of QTLs. These different methods have advantages and disadvantages to consider when selecting the appropriate model to be used, which is briefly discussed. (Source: Springer protocols...
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
Generation of Transgenic Rats Using Lentiviral Vectors
Transgenesis is a valuable tool with which to study different aspects of gene function in the context of the intact organism. During the last two decades a tremendous number of transgenic animals have been generated, and the continuous improvement of technology and the development of new systems have fostered their widespread application in biomedical research. Generally, transgenic animals are generated by introducing foreign DNA into fertilized oocytes, which can be achieved either by injecting recombinant DNA into the pronucleus or by transferring lentiviral particles into the perivitelline space. While mice remain the ...
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
DNA Vaccination Techniques
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...
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
Detection of Autoantibodies Against Myelin Oligodendrocyte Glycoprotein in Multiple Sclerosis and Related Diseases
We describe here in detail a sensitive cell-based assay that allows the identification of autoantibodies against MOG in serum. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
Methods for Ex Vivo Analysis of Immune Cell Function from the Central Nervous System
Experimental autoimmune encephalomyelitis (EAE) is an animal model commonly used to investigate the inflammatory response in organ-specific autoimmunity and a model of the early immune responses of multiple sclerosis. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
Neurofilament Light Chain Determination from Peripheral Blood Samples
The loss of neurological function is closely related to axonal damage. Neurofilament subunits are concentrated in neurons and axons and have emerged as promising biomarkers for neurodegeneration. Electrochemiluminescence (ECL) based assays are known to be of superior sensitivity and require less sample volume than conventional ELISAs. Here, we describe a highly sensitive ECL based immunoassay for quantification of neurofilament light chain (NfL) in blood and CSF. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - July 5, 2015 Category: Molecular Biology Source Type: news
High-Throughput Real-Time Analysis of Cell Oxygenation Using Intracellular Oxygen-Sensitive Probes
Knowledge of in situ oxygenation of cells in 2D and 3D cultures offers important insights into the impact of oxygen on cellular function. Here we outline how such measurements can be performed in 2D cultures of adherent cells and also in cells cultured on 3D scaffolds. Measurements are performed on conventional time-resolved fluorescence plate readers using the intracellular oxygen-sensitive probe MitoXpress®-Intra. We also illustrate how the impact of drug treatment on cell oxygenation can be assessed and how the link between oxygenation and glycolytic metabolism can be examined. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
Novel Methods for Measuring the Mitochondrial Membrane Potential
The mitochondrial membrane potential is a critical parameter for understanding mitochondrial function, but it is challenging to quantitate with current methodologies which are based on the accumulation of cation indicators. Recently we have introduced a new methodology based on the redox poise of the b-hemes of the bc
1 complex. Here we describe the thermodynamic framework and algorithms necessary to calculate the membrane potential from the measured oxidation states of the b-hemes. (Source: Springer protocols feed by Molecular Medicine)
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
Time-Resolved Spectrometry of Mitochondrial NAD(P)H Fluorescence and Its Applications for Evaluating the Oxidative State in Living Cells
Time-resolved fluorescence spectrometry is a highly valuable technological tool to detect and characterize mitochondrial metabolic oxidative changes by means of endogenous fluorescence (Chorvat and Chorvatova, Laser Phys Lett 6: 175–193, 2009). Here, we describe the detection and measurement of endogenous mitochondrial NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence directly in living cultured cells using fluorescence lifetime spectrometry imaging after excitation with 405 nm picosecond (ps) laser. Time-correlated single photon counting (TCSPC) method is employed. (Source: Springer protocols feed...
Source: Springer protocols feed by Molecular Medicine - January 1, 2015 Category: Molecular Biology Source Type: news
