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Membrane Translocation Assayed by Fluorescence Spectroscopy

Assessing the ability of biomolecules or drugs to overcome lipid membranes in a receptor-independent way is of great importance in both basic research and applications involving the use of liposomes. A combination of uptake, release, and dilution experiments performed by steady-sta ...

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Studying Lipid Organization in Biological Membranes Using Liposomes and EPR Spin Labeling

Electron paramagnetic resonance (EPR) spin-labeling methods provide a unique opportunity to determine the lateral organization of lipid bilayer membranes by discrimination of coexisting membrane domains or coexisting membrane phases. In some cases, coexisting membrane d ...

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The Use of Isothermal Titration Calorimetry to Study Multidrug Transport Proteins in Liposomes

Biophysical measurements of multidrug transporters in vitro can often be of limited relevance to the natural in vivo behavior. In particular, the properties of transporters when removed from their native bilayer and solubilized in detergents or lipids can differ significantly from t ...

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Methods to Monitor Liposome Fusion, Permeability, and Interaction with Cells

We describe fluorescence assays for membrane fusion involving the fusion of liposomes with each other and with cultured cells, fluorescence methods to assess liposome uptake by cells and the intracellular delivery of liposome contents, and assays to evaluate liposome membrane perm ...

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Assessment of LiposomeCell Interactions

Liposome-cell interactions have been assessed for over 30 years now by an enormous variety of approaches and methods. In-depth knowledge of liposome-cell interaction is still very relevant since new concepts and applications applying liposomes are being developed every day. This ch ...

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Studying Colloidal Aggregation Using Liposomes

Colloidal aggregation using liposomes has been studied in this chapter. As criteria of stability, the stability factor, an extension of the DLVO theory of colloidal stability, the fractal dimension of the liposome aggregates and the different regimes of aggregation (RLCA and DLCA) and the ...

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Use of Liposomes to Evaluate the Role of Membrane Interactions on Antioxidant Activity

Cellular membranes, which contain abundant phospholipids, such as phosphatidylcholine, are major targets subjected to the damage caused by free radicals. Cellular damage due to lipid oxidation is strongly associated with ageing, carcinogenesis and other diseases. In addition, ...

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Study of Respiratory Cytochromes in Liposomes

Important findings regarding the structure and function of respiratory cytochromes have been made from the study of these hemeproteins associated to liposomes. These studies contributed to the comprehension of the biological role of these proteins in the electron transfer proce ...

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Biosynthesis of Proteins Inside Liposomes

Protein expression is the most complex metabolic reaction that has been encapsulated in liposomes, mainly as an intermediate step toward the synthesis of minimal semisynthetic cells. Although there are different experimental approaches to achieving the synthesis of proteins in ...

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Visualization of Lipid Domain-Specific Protein Sorting in Giant Unilamellar Vesicles

Recent studies suggest that phospholipids in the plasma membrane of mammalian cells are not homogenously distributed but may form domains either by lipid-lipid interactions or/and as consequence of lipid-protein interactions. Such lipid compartments may act as protein recruit ...

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Fluorescence Correlation Spectroscopy for the Study of Membrane Dynamics and Organization in Giant Unilamellar Vesicles

Fluorescence correlation spectroscopy (FCS) is a powerful technique to study the lateral organization of membranes. It measures fluorescence intensity fluctuations in the single molecule regime and allows the determination of diffusion coefficients. When applied to lipid m ...

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Culture and Preparation of Human Embryonic Stem Cells for Proteomics-Based Applications

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 f ...

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A Two- and Three-Dimensional Approach for Visualizing Human Embryonic Stem Cell Differentiation

Undifferentiated human embryonic stem cells are characterized by expression of specific cell markers like the transcription factors OCT4, SOX2, and NANOG, the stage-specific embryonic antigen SSEA4, and the tumor-related antigens TRA-1-60 and TRA-1-81 and by their ability to diffe ...

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Immunoflourescence and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Grown Under Feeder-Free Conditions

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 t ...

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hESC Engineering by Integrase-Mediated Chromosomal Targeting

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 in ...

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Study of Gap Junctions in Human Embryonic Stem Cells

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 ...

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Human Embryonic Stem Cell Differentiation on Periodontal Ligament Fibroblasts

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 ...

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Generation of Neural Crest Cells and Peripheral Sensory Neurons from Human Embryonic Stem Cells

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 cent ...

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In Vitro Derivation of Chondrogenic Cells from Human Embryonic Stem Cells

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 obta ...

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Vascular Differentiation of Human Embryonic Stem Cells in Bioactive Hydrogel-Based Scaffolds

The vascularization of tissue constructs remains a major challenge in regenerative medicine, as the diffusional supply of oxygen can support only 100–200μm thick layers of viable tissue. The formation of a mature and functional vascular network requires communication between end ...

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