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This protocol describes a mass spectrometry–based workflow for combined analysis of protein phosporylation and N-glycosylation of extracellular vesicles obtained from a single blood plasma sample.
In this Protocol Extension, the authors extend their original Protocol used to generate a genome-scale metabolic model for a single strain to enable multi-strain models to be made, which can be used to study pan-metabolic capabilities and strain-specific differences across a species.
Tumor-reactive T cells are generated by coculturing tumor organoids and autologous peripheral blood lymphocytes and are evaluated for their capacity to carry out effector functions after recognition of tumor cells and whether they kill tumor organoids.
This protocol describes a collection of pH-sensitive fluorescent reporters that can be used for real-time dual-color imaging of exosome release from single cells. The authors provide detailed instructions for TIRF imaging and automated data analysis.
This Protocol describes a low-cost and scalable solid-state nanopore fabrication method, termed controlled breakdown (CBD), for fabricating solid-state nanopores.
G protein–coupled receptors (GPCRs) play a central role in physiological processes and are common drug targets. This GPCR precrystallization screen uses small culture volumes to determine which conditions result in maximal protein expression and stability.
DNA has the capacity to store large amounts of information for very long durations. This protocol describes encoding of digital files as DNA and the error-free retrieval of the stored data from the sequenced data.
Bacterial extracellular vesicles (BEVs) in human body fluids are analyzed using ultrafiltration, size-exclusion chromatography and density-gradient centrifugation to separate the BEVs, followed by post-separation characterization with orthogonal biochemical methods.
Transparent organs are obtained, with retained fluorescent protein signals, upon clearing by immersion in the appropriate CUBIC reagent. The positions of all the cells can be determined using the described software.
PheCAP takes structured data and narrative notes from electronic medical records and enables patients with a particular clinical phenotype to be identified.
This protocol covers the preparation and use of artificial microRNA clusters for gene therapy. The in silico design of chimeric microRNA transgenes in which up to six natural microRNA hairpins are replaced by synthetic versions obviates the need for complex molecular cloning.
hPSCs are differentiated into anterior foregut endoderm and then undergo lineage specification into NKX2-1+ lung progenitor cells. Next, the progenitors undergo distal/alveolar differentiation to produce self-renewing alveolar epithelial type II cells.
This protocol describes how to redesign, characterize, validate and apply genetically encoded dopamine sensors, such as those of the dLight1 family, to measure dopamine transients in cultured cells, neurons, acute brain slices and freely behaving, awake mice.
ICeChIP (internally calibrated ChIP) uses spiked-in, defined nucleosomal standards to overcome the pitfalls of traditional ChIP experiments, enabling the measurement of antibody specificity and the absolute measurement of histone modification density at genomic loci.
This protocol describes how to achieve high spatial resolution imaging of biological tissues using nanospray desorption electrospray ionization mass spectrometry
Surgical techniques are presented that facilitate decellularization of various mouse tissues. Immunostaining and microscopy of the resulting tissues is also presented using an extracellular matrix–specific antibody catalog.
The authors present a new protocol to quantitatively assess protein turnover in mice in vivo. The procedure includes instructions on the mouse diet for stable isotope labeling of amino acids in mammals (SILAM), sample preparation, mass spectrometry analysis and data processing.
This protocol provides instructions for the fabrication, assembly and operation of a microfluidic device used for chromatin immunoprecipitation followed by sequencing to profile histone modifications in low-input samples.