Springer Protocols platform has migrated to Experiments

Time 2020-09-17 10:03:26

Web Name: Springer Protocols platform has migrated to Experiments

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Antibody BETA Covering top cited antibodies from a curated subset of protocols Precise control of the gene copy number in the model yeast Saccharomyces cerevisiae may facilitate elucidation of enzyme functions or, in cell factory design, can be used to optimize production of proteins and metabolites. Currently, available…more Precise control of the gene copy number in the model yeast Saccharomyces cerevisiae may facilitate elucidation of enzyme functions or, in cell factory design, can be used to optimize production of proteins and metabolites. Currently, available methods can provide high gene-expression levels but fail to achieve accurate gene dosage. Moreover, strains generated using these methods often suffer from genetic instability resulting in loss of gene copies during prolonged cultivation. Here we present a method, CASCADE, which enables construction of strains with defined gene copy number. With our present system, gene(s) of interest can be amplified up to nine copies, but the upper copy limit of the system can be expanded. Importantly, the resulting strains can be stably propagated in selection-free media. less Techniques: Agarose Gel Electrophoresis, Gene Targeting, Metabolic Engineering, Yeast Transformation, PCR Ceramide can be generated on cell surfaces by the activity of the acid sphingomyelinase. The unique biophysical properties of ceramide result in the self-formation of small ceramide-enriched membrane domains that spontaneously fuse to large…more Ceramide can be generated on cell surfaces by the activity of the acid sphingomyelinase. The unique biophysical properties of ceramide result in the self-formation of small ceramide-enriched membrane domains that spontaneously fuse to large ceramide-enriched membrane macrodomains. The present chapter describes how these domains can be labeled and thereby visualized in cells. Further, the chapter provides protocols how ceramide and sphingosine can be quantified on the surface of cells and organs. less Techniques: Cell And Tissue Culture, Confocal Microscopy, Microscopy, Radioactive Labeling, Immunolabeling... 2 more Techniques: Cell And Tissue Culture, Confocal Microscopy, Microscopy, Radioactive Labeling, Immunolabeling, Thin-layer Chromatography, Specific Labeling less In DNA-based therapy research, the conception of a suitable vector to promote the target gene carriage, protection, and delivery to the cell is imperative. Exploring the interactions between polyethylenimine (PEI) and a plasmid DNA can give rise to…more In DNA-based therapy research, the conception of a suitable vector to promote the target gene carriage, protection, and delivery to the cell is imperative. Exploring the interactions between polyethylenimine (PEI) and a plasmid DNA can give rise to the formation of suitable complexes for gene release and concomitant protein production. The nanosystems formulation method, based on coprecipitation, seems to be adequate for the conception of nanoparticles with suitable properties (morphology, size, surface charge, and pDNA complexation capacity) for intracellular applications. The developed systems are able of cell uptake, intracellular trafficking, and gene expression, in an extent depending on the ratio of nitrogen to phosphate groups (N/P). It comes that the transfection process can be tailored by this parameter and, therefore, also the therapeutic outcomes. This knowledge contributes for progresses in the development of suitable delivery systems with potential application in DNA vaccines field. less Techniques: FTIR Spectroscopy, Agarose Gel Electrophoresis, Confocal Microscopy, Degassing, Transfection... 1 more Techniques: FTIR Spectroscopy, Agarose Gel Electrophoresis, Confocal Microscopy, Degassing, Transfection, Co-precipitation less RNA editing activity can be exploited for the restoration of disease-causing nonsense and missense mutations and as a tool to manipulate the transcriptome in a simple and programmable way. The general concept is called site-directed RNA editing and…more RNA editing activity can be exploited for the restoration of disease-causing nonsense and missense mutations and as a tool to manipulate the transcriptome in a simple and programmable way. The general concept is called site-directed RNA editing and has high potential for translation into the clinics. Due to its different mode of action RNA editing may well complement gene editing and other gene therapy options. In this method chapter, we particularly highlight RNA editing strategies that harness endogenous ADARs. Such strategies circumvent the delivery and expression of engineered editases and are notably precise and simple. This is particularly true if endogenous ADARs are recruited with chemically modified antisense oligonucleotides, an approach we call RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing). To foster the research and development of RESTORE we now report a detailed protocol for the procedure of editing reactions, and a protocol for the generation of partly chemically modified RESTORE ASOs with a combination of in vitro transcription and ligation. less Techniques: PAGE, CDNA Amplification, Cell And Tissue Culture, In Vitro Transcription, Transfection... 2 more Techniques: PAGE, CDNA Amplification, Cell And Tissue Culture, In Vitro Transcription, Transfection, PCR, HPLC less The RNA fluorescence in situ hybridization (RNA-FISH) methodology offers an attractive strategy to deepen our knowledge on the long noncoding RNA biology. In this chapter, we provide a comprehensive overview of the current RNA-FISH protocols…more The RNA fluorescence in situ hybridization (RNA-FISH) methodology offers an attractive strategy to deepen our knowledge on the long noncoding RNA biology. In this chapter, we provide a comprehensive overview of the current RNA-FISH protocols available for imaging nuclear and cytoplasmic lncRNAs within cells or tissues. We describe a multicolor approach optimized for the simultaneous visualization of these transcripts with their specific molecular interactors, such as proteins or DNA sequences. Common challenges faced by this methodology such as cell-type specific permeabilization, target accessibility, image acquisition, and post-acquisition analyses are also discussed. less Techniques: Agarose Gel Electrophoresis, Cell And Tissue Culture, FISH, In Situ Hybridization, Light Microscopy... 9 more Techniques: Agarose Gel Electrophoresis, Cell And Tissue Culture, FISH, In Situ Hybridization, Light Microscopy, Immunofluorescence, Image Processing, ImageJ, MetaMorph, Surface Rendering, Enzymatic Amplification, Bright-field Microscopy, Freeze-thaw Method, Cryosectioning less Polypeptide assembly and aggregation are the common forms of its physiological and pathological activity, and monitoring them on a molecular level is critical for resolving numerous medical (e.g., onset of neurodegenerative diseases) or biological…more Polypeptide assembly and aggregation are the common forms of its physiological and pathological activity, and monitoring them on a molecular level is critical for resolving numerous medical (e.g., onset of neurodegenerative diseases) or biological problems. Sensitivity of the intrinsic fluorescence of protein to its assembly, aggregation, or complexation offers a noninvasive methodology for identifying and determining different stages of these processes. In this protocol, we present the approach based on the time-resolved emission spectra (TRES), which reveals the number of fluorescent residues, the presence of dielectric relaxation, and the changes in fluorescence kinetics during aggregation. less Techniques: MALDI-TOF Mass Spectrometry, HPLC, Time-correlated Single Photon Counting Bioprinting has emerged as a promising method for precise spatiotemporal patterning of biological materials such as living cells, genetic materials, and proteins, which are sensitive to any other fabrication techniques. Bioprinting allows the…more Bioprinting has emerged as a promising method for precise spatiotemporal patterning of biological materials such as living cells, genetic materials, and proteins, which are sensitive to any other fabrication techniques. Bioprinting allows the generation of tissue constructs and models that closely mimic the anatomical and physiological attributes of a chosen tissue. In vitro toxicology assays can greatly benefit from bioprinting as drugs can be screened with higher efficiencies in a significantly reduced period. This protocol describes a method for fabricating bioprinted cartilage constructs which can be used for in vitro toxicology studies employing a scalable “tissue strand” bioprinting modality. less Techniques: Cell And Tissue Culture, Extrusion, Cell Proliferation Assay, Sterilization, Immunohistochemistry... 4 more Techniques: Cell And Tissue Culture, Extrusion, Cell Proliferation Assay, Sterilization, Immunohistochemistry, ImageJ, Cross-linking, Real-time PCR, Total RNA Extraction less Primordial germ cells (PGCs) are common ancestors of all germline cells. In mammals, PGCs emerge in early-stage embryos around the timing of gastrulation at or near epiblast, and specification of PGCs from their precursor cells involves multiple…more Primordial germ cells (PGCs) are common ancestors of all germline cells. In mammals, PGCs emerge in early-stage embryos around the timing of gastrulation at or near epiblast, and specification of PGCs from their precursor cells involves multiple growth factors secreted by adjacent cells. Recent advancements in germline stem cell biology have made it possible to generate PGC-like cell culture models (PGCLCs for PGC-like cells) from human and mouse pluripotent stem cells by mimicking the embryonic growth factor environment in vitro. Here we describe a method of producing human PGCLCs from primed-pluripotency induced pluripotent stem cells (iPSCs) via temporal conversion to naive pluripotency followed by formation of embryoid bodies (EBs) using the spin-EB method. less Techniques: Cell And Tissue Culture, Flow Cytometry, FACS, Immunostaining, Immunohistochemistry... 2 more Techniques: Cell And Tissue Culture, Flow Cytometry, FACS, Immunostaining, Immunohistochemistry, Phase Contrast Microscopy, Microdissection less Beyond cell proliferation, one of the most outstanding characteristics of the cancerous cells that promotes the tumoral progression is their high capacity to migrate and invade the surrounding healthy tissue. These cellular processes (migration and…more Beyond cell proliferation, one of the most outstanding characteristics of the cancerous cells that promotes the tumoral progression is their high capacity to migrate and invade the surrounding healthy tissue. These cellular processes (migration and invasion) are critical steps to metastasis. Metastatic progression of the tumors is often the leading cause of morbidity and mortality in cancer patients. Critical genes and cell signaling pathways involved in cell migration and invasion of tumor cells have been identified, and several clinical efforts to alleviate cancer are focused on them; however, once the tumor has metastasized, it is extremely difficult to stop the progression of very aggressive forms of cancer such as glioblastomas. Therefore, it is crucial to elucidate the specific molecular mechanisms underlying tumor progression. In this chapter, we describe some methods to study tumor progression by assessing migration and cell invasion in 2D and 3D cell culture conditions. less Techniques: 3D Cell Culture, Dissection, Cell Migration Assay, ImageJ, Cell Invasion Assay During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout …more During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization. less Techniques: Cell And Tissue Culture, Microscopy, Immunofluorescence, Cell Staining, Angiogenesis Assay Participate in User Experience Research Shape the way experiments are conducted in industrial R&D. 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