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Culture and Preparation of Human Embryonic Stem Cells for Proteomics-Based ApplicationsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
New challenges will arise as research into human embryonic stem (hES) cell differentiation moves from optimization and overcoming technical hurdles to mechanistic considerations. An immediate need will be to culture hES cells in the absence of contaminating feeder layers and allow for the preparation of purified DNA, RNA, and proteins to analyze changes in microRNA levels, gene expression, protein expression, and signal transduction. Purified, uniform populations of hES cells will allow researchers to better explore the biochemical mechanisms by which differentiation occurs. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Monitoring Stemness in Long-Term hESC Cultures by Real-Time PCREmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Human embryonic stem cells (hESC) involve long-term cultures that must remain undifferentiated. The real-time PCR (RT-PCR) technique allows the relative quantification of target genes, including undifferentiation and differentiation markers when referred to a housekeeping control with the addition of a calibrator that serves as an internal control to compare different lots of reactions during the time. The main aspects will include a minimal number of cells to be analyzed, genes to be tested, and how to choose the appropriate calibrator sample and the reference gene. In this chapter, we present how to apply the RT-PCR tech...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Single Cell Enzymatic Dissociation of Human Embryonic Stem Cells: A Straightforward, Robust, and Standardized Culture MethodEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The routine culture and expansion of human embryonic stem (hES) cells has been and is still posing a challenge to researchers wishing to take advantage of the cells' unique potential. In contrast to mouse embryonic stem cells, hES cells usually have to be expanded by tedious mechanical microdissection or by enzymatic dissociation to cell clusters of a very narrow size range. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Serum-Free and Feeder-Free Culture Expansion of Human Embryonic Stem CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Human embryonic stem cells (hESCs) are pluripotent stem cells derived from the inner cell mass of human blastocysts. hESCs have become a great asset to studying human diseases and genetic functions of healthy organisms. The rate at which hESCs are being used in laboratories is exponentially increasing, and with that, the need for xeno-free hESCs is also increasing. Xeno-free grade hESCs, cells that have not come into contact with any animal-derived components except those of human origin, are critical for eventual drug therapy, cell therapy, and disease treatment in humans. However, advances toward a xeno-free hESC environ...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Optimization of Physiological Xenofree Molecularly Defined Media and Matrices to Maintain Human Embryonic Stem Cell PluripotencyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe in this chapter the development of a xenofree molecularly defined medium, SBX, associated with xenofree matrices, to maintain human embryonic stem cell (hESC) pluripotency as determined by phenotypic, functional and TLDA studies. This simple, inexpensive, and more physiological culture condition has been chosen because (1) it is xenofree and molecularly defined; it is devoid of albumin, which is a carrier of undefined molecules; (2) it maintains pluripotency, but very significantly reduces differentiation gene expression during hESC self-renewal, as compared to the widely used culture conditions tested so far; ...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

In Vitro Neural Differentiation of Human Embryonic Stem Cells Using a Low-Density Mouse Embryonic Fibroblast Feeder ProtocolEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Human embryonic stem cells (hESCs) have the capacity to self-renew and to differentiate into all components of the embryonic germ layers (ectoderm, mesoderm, endoderm) and subsequently all cell types that comprise human tissues. HESCs can potentially provide an extraordinary source of cells for tissue engineering and great insight into early embryonic development. Much attention has been given to the possibility that hESCs and their derivatives may someday play major roles in the study of the development, disease therapeutics, and repair of injuries to the central and peripheral nervous systems. This tantalizing promise wi...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Growth of Human Embryonic Stem Cells Using Derivates of Human FibroblastsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The majority of human embryonic stem cell (hESC) lines have been derived and grown using mouse or human feeder cells, or using Matrigel®, an animal derivative rich in extracellular matrix (ECM) proteins. However, reliance on feeder layers and animal products limits the manipulation and clinical application of hESC. Alternatively, human fibroblasts produce an ECM which could be employed to coated plates and be easily sterilized. We have shown that hESC grown on this matrix and in the presence of medium conditioned by fibroblast cells maintain markers of pluripotency, including expression of cell surface proteins (SSEA3,...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Derivation of Human Embryonic Stem Cell Lines from Vitrified Human EmbryosEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here methods required to derive new embryonic stem cell lines starting from the initial cryopreservation of an embryo and finishing with a new cell line. We cover embryo cryopreservation and warming using a highly efficient vitrification method, the production of feeder cells and feeder plates, as well as embryo handling, plating and critical early passages, including earliest possible cryopreservation of putative stem cells using vitrification. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Human-Induced Pluripotent Stem Cells: Derivation, Propagation, and Freezing in Serum- and Feeder Layer-Free Culture ConditionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The recent discovery of genomic reprogramming of human somatic cells to an embryonic stem (ES) cell-like pluripotent state provides a unique opportunity for stem cell research. The reprogrammed cells, named as induced pluripotent stem (iPS) cells, possess many of the properties of ES cells and represent one of the most promising sources of patient-specific cells for use in disease model, development of pharmacology and toxicology, screening teratogens, and regenerative medicine. Here we describe the detailed methods for the generation of undifferentiated human iPS (hiPS) cells in feeder layer- and serum-free conditions. Th...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Genetic Manipulation of Human Embryonic Stem Cells in Serum and Feeder-Free MediaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Generic methods for genetic manipulation of human embryonic stem cells (hESCs) are important for both present research and future commercial applications. To date, differences in cell derivation and culture have required independent optimization of transfection and transduction protocols and some lines have remained refractile to all methods. Here we describe a culture protocol that has been extensively tested in 12 different hESC lines (1, 2) and shown to support efficient gene transfer independent of the method of gene delivery or history of the cell line. The system is based on Matrigel monolayer culture and conditioned...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Genetic Manipulation of Human Embryonic Stem CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
One of the great advantages of embryonic stem (ES) cells over other cell types is their accessibility to genetic manipulation. They can easily undergo genetic modifications while remaining pluripotent and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Since the first isolation of ES cells in mice, many effective techniques have been developed for gene delivery and manipulation of ES cells. These include transfection, electroporation, and infection protocols, as well as different approaches for inserting, deleting, or changing the expression of genes. These methods prov...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Transplantation of Human Embryonic Stem Cells and Derivatives to the Chick EmbryoEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Traditional methods of studying the differentiation of human embryonic stem cells (hESCs) include generation of embryoid bodies, induced differentiation in vitro, and transplantation to immune-deficient mice. The chick embryo is a well-studied and accessible experimental system that has been used for many years as a xenograft host for mammalian cells. Several years ago, we performed experiments transplanting colonies of hESC into organogenesis-stage chick embryos to establish a novel system for studying the developmental programs and decisions of pluripotent human cells. Fluorescent hESC were used, in order to permit ident...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Human Embryo Culture and Assessment for the Derivation of Embryonic Stem Cells (ESC)Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The culture and critical assessment of early human embryos during the first week of human development are reviewed for the derivation of ESC. Both normal and abnormal features are assessed by phase contrast microscopy of whole embryos and in serial sections of fixed material by light and electron microscopy (TEM). Normal embryos follow a time table of development and have equal blastomeres with minimal fragmentation and nuclear defects. Abnormal embryos show more fragmentation and nuclear aberrations such as micronucleation and multinucleation, reflected by aneuploidy, polyploidy, and mosaicism. The selection of normal emb...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Differentiation of Neural Precursors and Dopaminergic Neurons from Human Embryonic Stem CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here a reproducible, chemically defined protocol that allows directed differentiation of hESCs to nearly pure neuroectodermal cells and neurons. First, hESC colonies are detached from mouse fibroblast feeder layers and form aggregates to initiate the differentiation procedure. Second, after 4 days of suspension culture, the ESC growth medium is replaced with neural induction medium to guide neuroectodermal specification. Third, the differentiating hESC aggregates are attached onto the culture surface at day 6–7, where columnar neural epithelial cells appear and organize into rosettes. Fourth, the neural r...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Vascular Differentiation of Human Embryonic Stem Cells in Bioactive Hydrogel-Based ScaffoldsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The vascularization of tissue constructs remains a major challenge in regenerative medicine, as the diffusional supply of oxygen can support only 100–200 μm thick layers of viable tissue. The formation of a mature and functional vascular network requires communication between endothelial cells (ECs) and smooth muscle cells (SMCs). Potential sources of these cells that involve noninvasive methodologies are required for numerous applications including tissue-engineered vascular grafts, myocardial ischemia, wound healing, plastic surgery, and general tissue-engineering applications. Human embryonic stem cells (h...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

In Vitro Derivation of Chondrogenic Cells from Human Embryonic Stem CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Human embryonic stem cells (hESCs) have the ability to self-renew and differentiate into any cell lineage of the three germ layers, therefore holding great promise for regenerative medicine applications. However, directing lineage-restricted differentiation of hESCs and obtaining a homogenous differentiated cell population is still a challenge. We previously described a micromass culture system as a model system to study chondrogenic commitment of the hESCs. Using this system, various growth factors including BMP2 and TGFβ1 direct chondrogenic differentiation and modulate cartilage-specific matrix gene expression in a...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Embryonic Stem Cells as a Model for Studying Melanocyte DevelopmentEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Melanocytes are neural crest-derived pigment-producing cells that reside in the inner ear, in the uveal tract, in hair follicles, and in the skin. The main function of melanocytes is to provide pigmentation through melanin production and secretion to the immediate surrounding area. Although much is known about mature melanocyte function and regulation, particularly in the skin, little is known with regard to the signals and gene expression patterns that ensue upon melanocyte development and differentiation from embryonic precursors. The ability to examine these patterns in an in vitro specified setting through the use of e...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Generation of Neural Crest Cells and Peripheral Sensory Neurons from Human Embryonic Stem CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Peripheral somatic sensory neurons (PSNs) are responsible for the critical function of transmitting multiple modalities of information from the outside world, including heat, touch, and pain, as well as the position of muscles required for coordinated voluntary movement to the central nervous system. Many peripheral neuropathies exist, including hereditary neurodegeneration in Familial Dysautonomia, infections of PSNs by viruses such as Varicella zoster and damage to PSNs and/or their process resulting from other disease conditions such as diabetes. Understanding of the etiology of these diseases and development of treatme...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Human Embryonic Stem Cell Differentiation on Periodontal Ligament FibroblastsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types made them potential cell source in different cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering aims to improve the outcome of regenerative therapies which have variable success rates when contemporary techniques are used. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with guided tissue-regenerat...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

hESC Engineering by Integrase-Mediated Chromosomal TargetingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Bacteriophage recombinases can target specific loci in human embryonic stem cells (hESCs) at high efficiency allowing for long-term expression of transgenes. In this chapter, we describe a retargeting system where phiC31 integrase is used to deliver a chromosomal target for a second integrase, R4. The engineered hESC line can be adapted for complex element assembly using Multisite Gateway technology. Retargeted clones show sustained expression and appropriate regulation of the transgenes over long-term culture and upon differentiation. The system described here represents a method to rapidly assemble complex plasmid-based ...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Study of Gap Junctions in Human Embryonic Stem CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Gap junctional intercellular communication (GJIC) has been described in different cell types including stem cells and has been involved in different biological events. GJIC is required for mouse embryonic stem cell maintenance and proliferation, and various studies suggest that functional GJIC is a common characteristic of human embryonic stem cells (hESC) maintained in different culture conditions. This chapter introduces methods to study gap junctions in hESC, from expression of gap junction proteins to functional study of GJIC in hESC proliferation, apoptosis, colony growth, and pluripotency. (Source: Springer protocols...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Immunoflourescence and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Grown Under Feeder-Free ConditionsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This chapter describes the procedures in order to do immunofluorescence (IF) microscopy and quantitative PCR (qPCR) analysis of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol outlining the steps from initially growing the cells, passaging onto 16-well glass chambers, and continuing with the general IF and qPCR steps will be provided. The techniques will be illustrated with new results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, and PDGF signaling pathways to primary cilia in stem cell maintenance and differentiation. Furth...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

A Two- and Three-Dimensional Approach for Visualizing Human Embryonic Stem Cell DifferentiationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe a technique allowing paraffin embedding an entire hESC colony (e.g., ˜ 150 µm thick) and prepare 2-µm thick serial sections. Different staining procedures applied to individual sections produce a 2D survey of the developing hESC colony. Furthermore, a new and useful visualization of this 2D-expression pattern can be created by developing a 3D-model of the culture, based on serial paraffin sections. Individual sections are stained using individual markers. Using 3D image processing software such as Mimics or 3D-Doctor, the actual 3D-rendering of an entire colony can be accomplished. An extended...
Source: Springer protocols feed by Cell Biology - October 13, 2009 Category: Cytology Source Type: info

Scanning for Transcription Factor Binding by a Variant EMSAEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe here our use and refinement of a variant EMSA that can employ multiple and relatively long (up to 1000 bp) probes of promoter sequence in one binding reaction for interaction with nuclear proteins in general and individual transcription factors in particular. We provide labeling and electrophoresis methods suitable for such probes and also highlight the mobility shift differences one can expect with the variant probe method. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 7, 2009 Category: Cytology Source Type: info

RNA Interference in Keratinocytes and an Organotypic Model of Human EpidermisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Gene silencing approaches afford investigators the ability to gain important insight into the normal functional requirements of specific epidermal proteins and promise to yield a powerful therapeutic means to dampen the level of proteins that are mutated or frequently overexpressed in skin disease. The efficient and tractable delivery of siRNAs into epidermal keratinocytes is seminal to this process. Here, we describe techniques for transient and long-term silencing of a representative gene product, namely desmoglein 1, in primary human epidermal keratinocytes maintained as submerged cultures or three-dimensional organotyp...
Source: Springer protocols feed by Cell Biology - October 7, 2009 Category: Cytology Source Type: info

F2-Isoprostanes: Sensitive Biomarkers of Oxidative Stress In Vitro and In Vivo: A Gas Chromatography-Mass Spectrometric ApproachEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A gas chromatography-mass spectrometric method was developed that allowed the accurate, highly sensitive and specific quantification of F2-isoprostanes (F2-IsoPs) in different tissues and body fluids. Measurement of F2-IsoPs in isolated rat brain mitochondria, HaCaT keratinocytes, human plasma, and microdialysates of human skin has established the occurrence of oxidative stress in a variety of model systems and disease states. F2-IsoPs correlated with other markers of lipid peroxidation (e.g., TBARS, HETEs) in experimental models of oxidative stress. F2-IsoPs were elevated about 100-fold after iron/ascorbate-induced oxidat...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Quantification of Lysophosphatidylcholine Species by High-Throughput Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS)Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Lysophosphatidylcholine (LPC) is a bioactive lipid implicated to play a functional role in various diseases including atherosclerosis, diabetes, cancer, and inflammation. Conventional methods are of limited value for a systematic evaluation of LPC species concentrations due to complicated, time-consuming procedures. Here we describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC species quantification. This assay provides accuracy and precision sufficient for the analysis of large clinical studies as well as basic biochemical studies in a broad range o...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Volatile Oxylipins and Related Compounds Formed Under Stress in PlantsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Plants form volatile oxylipins and related compounds under stress. Some of them are important flavor chemicals and give big impact on the flavor quality of food made from plant materials. They are also involved in defense responses of plants against pathogens and herbivores. Furthermore, in some instances, they cause harmful effects on plants themselves. Because of these significances of volatile oxylipins and related compounds, demands to perform comprehensive analyses of these compounds are increasing. In this chapter, we describe the simple but efficient procedures to reveal profiles of volatile oxylipins and related co...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Use of Lipidomics for Analyzing Glycerolipid and Cholesteryl Ester Oxidation by Gas Chromatography, HPLC, and On-line MSEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Various analytical techniques have been adopted for the isolation and identification of the oxolipids and for determining their functionality. Gas chromatography in combination with mass spectrometry (MS) has been specifically utilized in analysis of isoprostanes and other low molecular weight oxolipids, although it requires derivatization of the solutes. In contrast, liquid chromatography (LC) in combination with on-line MS has proven to be well suited for analysis of intact oxolipids without (or minimal) derivatization. LC-MS has also been helpful for the identification of lipidomic changes resulting from covalent bindin...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Lipid Raft-Redox Signaling Platforms in Plasma MembraneEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Membrane lipid rafts (LRs) have been demonstrated to be importantly involved in transmembrane signaling in a variety of mammalian cells. Many receptors can be aggregated within the LR clusters to form signaling platforms. Currently, LRs were reported to be clustered to aggregate, recruit, and assemble NADPH oxidase subunits and related proteins in various cells in response to various stimuli, forming redox signaling platforms. These LR signaling platforms may play important roles in the regulation of cellular activity and cell function, and also in the development of cell dysfunction or injury associated with various patho...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Mass Spectrometry Analysis of Polyisoprenoids Alcohols and Carotenoids via ESI(Li+)-MS/MSEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Direct analysis of polyisoprenoid alcohols by electrospray ionization mass spectrometry (ESI-MS) often produces poor results requiring off-line time- and sample-consuming derivatization techniques. In this chapter, we describe a simple ESI-MS approach for the direct analysis of polyisoprenoid alcohols from biological samples. Lithium iodide is used to promote cationization by intense formation of [M+Li]+ adducts. Detection of polyisoprenoids with mass determination can thus be performed with high sensitivity (LOD near 100 pM), whereas characteristic collision-induced dissociations observed for both dolichols and polyprenol...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Detection of a Lipid-Lysine Adduct Family with an Amide Bond as the Linkage: Novel Markers for Lipid-Derived Protein ModificationsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
An amide-type adduct, hexanoyl-lysine (HEL) is generated from the reaction between n-6 fatty acid (FA)-derived lipid peroxide and lysine. Immunochemical and chemical methods can be used to detect the formation of HEL. For example, an ELISA kit using the monoclonal antibody to HEL is now commercially available. We recently identified propanoyl-lysine (propionyl-lysine, PRL) from the reaction of an n-3 FA and a lysine residue. The antibody to PRL has been prepared and characterized. Using these monoclonal antibodies, the localization of adducts in tissues has been confirmed. Moreover, both amide-type adducts, HEL and PRL, ca...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Mass-Spectrometric Characterization of Phospholipids and Their Hydroperoxide Derivatives In Vivo: Effects of Total Body IrradiationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe application of this methodology in assessments of phospholipid hydroperoxides using as an example their characterization and quantitative determinations in different tissues of mice exposed to total body irradiation (TBI, 10 and 15 Gy). Using ESI-MS, we identified individual molecular species – with particular emphasis on polyunsaturated molecules as preferred peroxidation substrates – in major classes of phospholipids: cardiolipin (CL), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) isolated from mouse brain, lung, muscles, small inte...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Assessing the Neuroprotective Effect of Antioxidative Food Factors by Application of Lipid-Derived Dopamine Modification AdductsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Advances in understanding the neurodegenerative pathologies are creating new opportunities for the development of neuroprotective therapies, such as antioxidant food factors, lifestyle modification, and drugs. However, the biomarker by which to determine the effect of the agent on neurodegeneration is limited. We here address hexanoyl dopamine (HED), one of novel dopamine adducts derived from brain polyunsaturated acid, referring to its in vitro formation, potent toxicity to SH-SY5Y cells, and application to assess the neuroprotective effect of antioxidative food factors. Dopamine is a neurotransmitter and its deficiency i...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Comprehensive mRNA Profiling of Lipid-Related Genes in Microglia and Macrophages Using Taqman ArraysEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Quantitative real-time reverse-transcription (RT)-PCR is a precise and sensitive method to measure mRNA levels over a broad dynamic range. This chapter describes the quantitative transcript analysis of 41 selected lipid-related transcripts in macrophages and microglia using a novel “Lipidomic” Taqman Array. The Taqman Array results show that (1) stimulation with the liver-X-receptor and retinoid-X-receptor ligands T0901317 and 9-cis retinoic acid induces several genes of lipid metabolism, (2) lipopolysaccharide (LPS) and interferon-g (Ifn-g) strongly repress lipid-related genes, and (3) coincubation with docosa...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Monitoring Sterol Uptake, Acetylation, and Export in YeastEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Sterols are essential lipid components of eukaryotic membranes. They are synthesized in the endoplasmatic reticulum (ER) from where they are efficiently transported to the plasma membrane, which harbors ~90% of the free sterol pool of the cell. The molecular mechanisms that govern this lipid transport, however, are not well characterized and are challenging to analyze. Saccharomyces cerevisiae offers the opportunity to circumvent some of the technical limitations associated with studying this forward transport of sterols from the ER to the plasma membrane, because the organism can also take up sterols from the environment,...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Imaging Lipid Membrane Domains with Lipid-Specific ProbesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Imaging membrane lipid domains to characterize their organization and function has been hindered by the lack of reliable lipid-specific probes. In this paper, we provide detailed methods to investigate, mainly by confocal microscopy, the distribution and dynamics of two components of the “lipid rafts,” sphingomyelin (SM) and cholesterol, using two specific lipid probes that have been extensively studied in the laboratory: lysenin, a SM-binding toxin and the fluorescent esters of poly(ethylene glycol) cholesteryl ether (PEG-Chol) that label cholesterol-rich domains. The production of nontoxic forms of lysenin as...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Methods to Monitor Fatty Acid Transport Proceeding Through Vectorial AcylationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The process of fatty acid transport across the plasma membrane occurs by several mechanisms that involve distinct membrane-bound and membrane-associated proteins and enzymes. Among these are the fatty acid transport proteins (FATP) and long-chain acyl CoA synthetases (Acsl). Previous studies in yeast and adipocytes have shown FATP and Acsl form a physical complex at the plasma membrane and are required for fatty acid transport, which proceeds through a coupled process-linking transport with metabolic activation termed vectorial acylation. At present, six isoforms of FATP and five isoforms of ACSL have been identified in mi...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Activity-Based Profiling of Lipases in Living CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The ultimate goal of proteomics is to characterize the function of all proteins in parallel and in the most physiologically relevant settings possible. A step toward this goal has been the introduction of activity-based proteomics. The simultaneous detection of individual protein activities can be facilitated directly in the proteome using specific activity-based probes consisting of a recognition site targeting a certain enzyme species, a properly positioned reactive site which forms a covalent bond with the target and a reporter tag for visualization and/or purification of the covalently bound target. As properties like ...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Histochemistry and Lipid Profiling Combine for Insights into Aging and Age-Related MaculopathyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Aging is the major risk factor for age-related maculopathy (ARM), the biggest cause of vision loss among the elderly in industrialized societies, and a major change in the affected tissues is the age-related accumulation of neutral lipid in Bruch’s membrane (BrM) of the eye throughout adulthood. Here we show that esterified cholesterol (EC) is the major neutral lipid species in this tissue, which has implications for potential sources of this material. The combination of filipin histochemistry and comprehensive lipid profiling made possible this insight on a complex tissue. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Instrument-Independent Software Tools for the Analysis of MS–MS and LC–MS Lipidomics DataEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Mass spectrometry (MS), particularly electrospray-MS, is the key tool in modern lipidomics. However, as even a modest scale experiment produces a great amount of data, data processing often becomes limiting. Notably, the software provided with MS instruments are not well suited for quantitative analysis of lipidomes because of the great variety of species present and complexities in response calibration. Here we describe the use of two recently introduced software tools: lipid mass spectrum analysis (LIMSA) and spectrum extraction from chromatographic data (SECD), which significantly increase the speed and reliability of m...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Computer-Assisted Interpretation of Triacylglycerols Mass SpectraEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Triacylglycerols (TGs) are principal components of vegetable oils and animal fats. Natural TGs form extremely complex mixtures composed of tens or hundreds of molecular species. HPLC/MS suits well for their analyses, but manual data processing is laborious and time-consuming. Specialized software algorithms are needed to accelerate the interpretation process. Here we present software named TriglyAPCI for interpreting APCI, APPI, or ESI MS/MS spectra of TGs. The chapter shows how to build and use the software, what are its advantages and limitations. The algorithm uses diacylglycerol fragments and molecular adducts for dete...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Visualization of Complex Processes in Lipid Systems Using Computer Simulations and Molecular GraphicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Computer simulation has become an increasingly popular tool in the study of lipid membranes, complementing experimental techniques by providing information on structure and dynamics at high spatial and temporal resolution. Molecular visualization is the most powerful way to represent the results of molecular simulations, and can be used to illustrate complex transformations of lipid aggregates more easily and more effectively than written text. In this chapter, we review some basic aspects of simulation methodologies commonly employed in the study of lipid membranes and we describe a few examples of complex phenomena that ...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Bioinformatics Strategies for the Analysis of LipidsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Owing to their importance in cellular physiology and pathology as well as to recent technological advances, the study of lipids has reemerged as a major research target. However, the structural diversity of lipids presents a number of analytical and informatics challenges. The field of lipidomics is a new postgenome discipline that aims to develop comprehensive methods for lipid analysis, necessitating concomitant developments in bioinformatics. The evolving research paradigm requires that new bioinformatics approaches accommodate genomic as well as high-level perspectives, integrating genome, protein, chemical and network...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

The Effect of Lipid Adjustment on the Analysis of Environmental Contaminants and the Outcome of Human Health RisksEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Past literature on exposure to lipophilic agents such as organochlorines (OCs) is conflicting, posing challenges for the interpretation of their potential human health risks. Since blood is often used as a proxy for adipose tissue, it is necessary to model serum lipids when assessing health risks of OCs. Using a simulation study, we evaluated four statistical models (unadjusted, standardized, adjusted, and two-stage) for the analysis of polychlorinated biphenyls (PCBs) exposure, serum lipids, and health outcome risk. Eight candidate true causal scenarios, depicted by directed acyclic graphs, were used to illustrate the ram...
Source: Springer protocols feed by Cell Biology - October 1, 2009 Category: Cytology Source Type: info

Mapping the Lipolytic Proteome of Adipose Tissue Using Fluorescent Suicide InhibitorsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Lipases are responsible for the hydrolysis of acylglycerols and cholesteryl esters in animals, plants, and microorganisms. In this chapter we describe a tool for the concomitant analysis of lipases in complex proteomes. For this purpose, the target enzymes are selectively and covalently labelled with fluorescent suicide inhibitors. Stable lipid–protein complexes are formed that are separated by gel electrophoresis and identified by mass spectrometry. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - September 22, 2009 Category: Cytology Source Type: info

Non-invasive Mapping of Lipids in Plant Tissue Using Magnetic Resonance ImagingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Plant oil has become an important component in the search for a replacement for non-renewable energy sources, as well as being used for a wide range of industrial purposes, all in addition to its vital importance for human diet. Detailed knowledge of the lipid distribution in plants is fundamental for the understanding of local regulatory networks covering storage metabolism, and for the development of new approaches for plant breeding and transgenic research. We here review a measurement protocol or “tool” based on magnetic resonance imaging (MRI), which allows the non-invasive detection and quantitative visua...
Source: Springer protocols feed by Cell Biology - September 22, 2009 Category: Cytology Source Type: info

Brain Phosphoinositide Extraction, Fractionation, and Analysis by MALDI-TOF MSEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) can provide rapid, sensitive determinations of lipids from small tissue samples in both single determinations and automated high-throughput assays. MALDI-TOF MS is a sensitive, high-throughput technique for the determination of lipids such as the phosphoinositides, PtdIns (phosphatidylinositol), PIP (phosphatidylinositol-4-phosphate, and PIP2 (phosphatidylinositol-4,5-bisphosphate), but in crude extracts the signals are weak or not observed due in large part to ion suppression by phosphatidylcholine and other cationic lipids. ...
Source: Springer protocols feed by Cell Biology - September 22, 2009 Category: Cytology Source Type: info

HPLC/MS/MS-Based Approaches for Detection and Quantification of EicosanoidsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Eicosanoids are oxygenated, endogenous, unsaturated fatty acids derived from arachidonic acid. Detection and quantification of these compounds are of great interest because they play important roles in a number of significant diseases, including asthma, chronic obstructive pulmonary disease (COPD), cardiovascular disease, and cancer. Because the endogenous levels of eicosanoids are quite low, sensitive and specific analytical methods are required to reliably quantify these compounds. High-performance liquid chromatography mass spectrometry (HPLC/MS) has emerged as one of the main techniques used in eicosanoid profiling. He...
Source: Springer protocols feed by Cell Biology - September 22, 2009 Category: Cytology Source Type: info

Lipidomics of the Red Cell in Diagnosis of Human DisordersEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Applications of tandem mass spectrometry in the field of lipid clinical chemistry are considered. Haemato­logical and biochemical advantages are presented favoring the choice of red blood cell membranes as a starting material in a wide variety of biomedical fields. Practical considerations are discussed with respect to methods of sampling, storage, and lipid extraction of red blood cells. The chapter describes the capabilities of a direct infusion of raw lipid extracts in the electro-spray ionization source compared with the more sophisticated method of high-performance liquid chromatography coupled with hybrid tandem ...
Source: Springer protocols feed by Cell Biology - September 22, 2009 Category: Cytology Source Type: info