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Multicolor labeling of cardiac muscle with systemic AAVs. Three fluorescent proteins (mTurquoise2, mNeonGreen, and mRuby2) were separately packaged into AAV-PHP.S and systemically codelivered to a wild-type mouse at 3.3 × 1011 vector genomes (vg) per virus (1 × 1012 vg total). Gene expression in the heart was evaluated 11 d later. Individual cardiomyocytes can be easily distinguished from one another.
This protocol describes the analysis of stable isotope (13C and 15N) incorporation into polar metabolites in central carbon metabolic pathways using HILIC separation and selected reaction monitoring with a hybrid triple quadrupole mass spectrometer.
This protocol describes how to inject therapeutic cells, viruses, or other macromolecular products into the mouse or rat lumbar ventral cord, modeling the routes used for administration of therapies currently in use in human clinical trials.
Stem cells with blastomere-like features are derived from single eight-cell-stage blastomeres or whole eight-cell pre-implantation mouse embryos, or by conversion of mouse ES or induced pluripotent stem (iPS) cells reprogrammed from fibroblasts.
Having developed AAV capsids that target sites throughout the body, here the authors describe how to produce and systemically administer these AAVs to rodents to label and/or genetically manipulate cells in the nervous system and visceral organs.
Multiplexed sequencing of barcoded mutant collections enables high-throughput, fitness-based condition profiling. BEAN-counter is a computational pipeline for quantifying mutant sensitivity or resistance for a few to thousands of conditions.
This protocol describes an approach for cell-type-specific detection of newly synthesized proteins in vivo. To this end, proteins are pulse-labeled with a puromycin analog (OP-Puro) and then undergo fluorescent labeling and flow cytometry analysis.
Focusing on the neglected tropical disease schistosomiasis, this protocol details how to establish and maintain the Schistosoma mansoni life cycle in the lab, culture relevant parasite stages and perform in vitro and in vivo drug-screening assays.
Human pluripotent stem cells are differentiated into ventral–anterior foregut spheroids and then to lung organoids, resembling the bronchi and surrounding mesenchyme of the developing human airway, or bud tip progenitor organoids.
Schwannoma cells are injected into the CPA region of the mouse brain to establish a model of vestibular schwannoma. Procedures for intravital imaging and neurological tests for hearing are also described.
This protocol describes a strategy for cell-specific labeling of nascent proteomes in vivo. Cell-type-specific proteins are tagged with a noncanonical amino acid in live mice and then are subjected to affinity purification and identification by MS.
This protocol describes how to fabricate, calibrate, and use micropipette force sensors for measurements in the sub-nanonewton to millinewton range. The micropipettes can be used on samples ranging from single cells to millimeter-sized organisms.
This protocol describes enhanced number and brightness (eN&B), an approach that uses fluorescence fluctuation spectroscopy data to directly measure the oligomerization state and dynamics of fluorescently tagged proteins in living cells.