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This protocol describes how to engraft human cancer cells in immunocompromised adult zebrafish. The fish are first adapted to 37 °C, followed by intraperitoneal or periocular muscle transplantation of xenograft cells and fluorescence imaging.
Here the authors describe a GUI-based protocol called FMAP for using funnel metadynamics to calculate the absolute binding free energy of a ligand to its molecular target and predict the ligand binding mode and mechanism.
This protocol describes how to engineer DNA nanostructures with different sizes, shapes and mechanical properties; load them with a siRNA cargo; and evaluate their ability to silence genes in mature tobacco plants.
This protocol describes the design and synthesis of CRISPR-responsive smart hydrogels and their actuation for both the controlled release of cargos (small molecules, enzymes, nanoparticles and living cells) and diagnostic applications.
This protocol describes a procedure for live-cell imaging of endocytic events in cultured cells using a pH-sensitive fluorophore and fast extracellular pH changes. A MATLAB-based analysis pipeline is provided to facilitate automated data processing.
The authors describe detailed procedures for an epigenomic profiling method suitable for low-input samples that is based on in situ labeling with an oligonucleotide-conjugated antibody.
GOTI (genome-wide off-target analysis by two-cell embryo injection) detects off-target mutations of CRISPR–Cas9-based genome editing and base editing. One blastomere of a two-cell mouse embryo is edited so that edited and unedited cells from the same genetic background can be compared.
This protocol describes a microfluidic platform for dynamic high-throughput analysis of the phenotypes of single cells. Cell-surface markers and secreted proteins are quantified and characterized by fluorescence detection using tailored immunoassays.
This protocol describes the MAC-tag approach, which combines affinity purification and biotinylation identification proximity labeling in a single tag. Binding proteins are identified by liquid chromatography–mass spectrometry, followed by visualization of protein localization using an online platform.
Embryonic stem cells undergo CRISPR–Cas9-mediated editing and are then used to reconstitute forebrain regions in mouse chimeras via neural blastocyst complementation.
Formalin fixation and paraffin embedding (FFPE) of human tissue is a central strategy for preserving pathological specimens. This protocol describes how to process these specimens for spatially resolved LC-MS by laser-capture microdissection.
This protocol provides standardized laboratory manufacturing practices to select, cultivate and purify bacteriophages for human clinical applications. The procedure covers all stages from phage isolation and characterization to quality control.
Proteomic cysteines can undergo redox reactions and electrophile-derived modifications. In QTRP, a thiol-reactive probe is used to covalently label, enrich and quantify the reactive cysteinome in cultured cells and tissue samples.
This protocol addresses the challenge of detecting and quantifying known protein complexes at the proteome level using SEC, DIA/SWATH mass spectrometry and a computational framework, CCprofiler.
This protocol describes the isolation and 3D culture of organoids from fresh murine or human primary and metastatic tumor tissue and provides instructions for real-time imaging, genetic and microenvironmental manipulation and molecular analysis.
This protocol describes a PB transposon-based mutagenesis system for construction of high-quality mutant libraries in haploid Candida species. The procedure describes mutant library generation, PB insertion-site sequencing and data analysis.
This protocol describes how to build and implant an in vivo compression device that simulates the solid mechanical forces exerted on the mouse brain by a growing tumor. Standard transparent cranial windows are adapted to include a turnable screw for controlled compression/decompression.
This protocol describes a workflow for site-specific extraction and mass spectrometry-based identification of O-linked glycopeptides (EXoO). The protocol can be applied to cultured cells, tissues and plasma.
Nicholson et al. describe a system for identifying molecular species derived from nuclear magnetic resonance spectroscopy-based metabolic phenotyping studies, with detailed information on sample preparation, data acquisition and modeling. They recommend eight modular workflows to be followed in sequential order.
Treatment with the TLR7/8 ligand (R848) precipitates X sperm, thus facilitating the separation of X sperm from Y sperm. The provided protocols for mouse and bovine sperm separation can then be used in IVF.