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Protocol
Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9
Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for...
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Protocol
Behavioral Tests for Associative Learning in Caenorhabditis elegans
Learning is critical for survival as it provides the capacity to adapt to a changing environment. At the molecular and cellular level, learning leads to alterations within neural circuits that include synaptic...
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Protocol
Long-Term Preservation of Plant Viruses in Cryopreserved Shoot Tips
Availability of the methods for long-term virus preservation facilitates easy acquirement of viruses, which are needed in many basic and applied virological studies. Cryopreservation is currently considered an...
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Protocol
13C-Fingerprinting and Metabolic Flux Analysis of Bacterial Metabolisms
13C-assisted metabolism analysis provides rigorous calculations of the intracellular reaction rates (i.e., fluxes) within the central metabolism of microbial hosts. This map** of the intracellular network with....
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Protocol
Ultra-Performance Liquid Chromatography/Mass Spectrometry for the Detection and Quantification of Diffusible Signal Factor (DSF) Family Quorum-Sensing Signals
Molecules of the diffusible signal factor (DSF) family represent a class of widely conserved quorum-sensing signals used by many Gram-negative bacterial pathogens. The measurement of DSF family signals is esse...
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Protocol
Monitoring the Integrity of the Blood-Testis Barrier (BTB): An In Vivo Assay
The blood-testis barrier is a unique ultrastructure in the mammalian testis, located near the basement membrane of the seminiferous tubule that segregates the seminiferous epithelium into the basal and the adl...
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Protocol
Fluorescence-Based Ratiometric Measurement of CRAC Channel Activity in STIM-Orai-Overexpressing HEK-293 Cells
Calcium influx through store-operated Ca2+ entry (SOCE), mediated by STIM-operated Orai channels, is crucial for many cellular functions. To dissect the molecular mechanisms underlying the process of STIM-Orai ac...
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Protocol
Engineered Cross-Linking to Study the Pore Architecture of the CRAC Channel
ORAI1 constitutes the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a prototypical store-operated channel that is essential for the activation of cells of the immune system. Her...
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Protocol
Integrated Multimodal Evaluation of Genotoxicity in ZFN-Modified Primary Human Cells
Iatrogenic adverse events in clinical trials of retroviral vector-mediated gene-corrected cells have prioritized the urgent need for more comprehensive and stringent assessment of potentially genotoxic off-tar...
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Protocol
A Mini-Electrochemical System with Integrated Micropipet Tip and Pencil Graphite Electrode for Measuring Cytotoxicity
A novel mini-electrochemical system has been developed for evaluating cytotoxicity of anticancer drugs based on trace cell samples. The mini-electrochemical system was integrated by using pencil graphite modif...
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Protocol
Directed Differentiation of Human-Induced Pluripotent Stem Cells to Mesenchymal Stem Cells
Multipotent stromal cells, also known as mesenchymal stem cells (MSCs), possess great potential to generate a wide range of cell types including endothelial cells, smooth muscle cells, bone, cartilage, and lip...
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Protocol
Quantitative Analysis of Human Cancer Cell Extravasation Using Intravital Imaging
Metastasis, or the spread of cancer cells from a primary tumor to distant sites, is the leading cause of cancer-associated death. Metastasis is a complex multi-step process comprised of invasion, intravasation...
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Protocol
Directed Endothelial Progenitor Differentiation from Human Pluripotent Stem Cells Via Wnt Activation Under Defined Conditions
Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development, disease modeling, drug discovery, and cell-based ...
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Protocol
Generation of Epithelial Cell Populations from Human Pluripotent Stem Cells Using a Small-Molecule Inhibitor of Src Family Kinases
Human pluripotent stem cells (hPSCs), under the right conditions, can be engineered to generate populations of any somatic cell type. Knowledge of what mechanisms govern differentiation towards a particular li...
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Protocol
Eosinophil Shape Change and Secretion
The analysis of eosinophil shape change and mediator secretion is a useful tool in understanding how eosinophils respond to immunological stimuli and chemotactic factors. Eosinophils undergo dramatic shape cha...
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Protocol
Map** of Ago2–GW182 Functional Interactions
MicroRNA (miRNA)-mediated posttranscriptional regulation of gene expression has become a major focus in understanding fine-tuning controls in many biological processes. Argonaute 2 protein (Ago2), a core compo...
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Protocol
Use of Stem Cell Radiation Chimeras to Analyze How Domains of Specific Proteins Impact on Murine NK Cell Development In Vivo
Although the use of mutant mice has been extremely useful in identifying those proteins and molecules specifically required for the development of NK cells, the establishment of a well-defined protocol to repl...
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Protocol
Automated Generic Analysis Tools for Protein Quantitation Using Stable Isotope Labeling
Isotope labeling combined with LC-MS/MS provides a robust platform for quantitative proteomics. Protein quantitation based on mass spectral data falls into two categories: one determined by MS/MS scans, e.g., ...
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Protocol
Subnuclear Localization and Intranuclear Trafficking of Transcription Factors
Nuclear microenvironments are architecturally organized subnuclear sites where the regulatory machinery for gene expression, replication, and repair resides. This compartmentalization is necessary to attain re...
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Protocol
Chromatin Immunoprecipitation Assays: Application of ChIP-on-Chip for Defining Dynamic Transcriptional Mechanisms in Bone Cells
Normal cell growth and differentiation of bone cells requires the sequential expression of cell type specific genes to permit lineage specification and development of cellular phenotypes. Transcriptional activ...
