Bio-protocol最新文献

Bottom-up tissue engineering using cell spheroids offers many advantages in recapitulating native cell-cell and cell-matrix interactions. Many tissues, such as cartilage, bone, cardiac muscle, intestine, and neural tissues, have been tissue-engineered using cell spheroids. However, previous methods for spheroid assembling, such as mold casting, hydrogel-based bioprinting, or needle array, either lack control over final tissue geometry or face challenges in scalability and throughput. In this protocol, we describe a robust and scalable tissue engineering method for assembling cell spheroids into a thin, planar spheroid sheet. The spheroids are sandwiched between two flexible meshes held by a frame, facilitating uniform spheroid fusion while ensuring nutrient exchange and ease of handling. We demonstrate this method by producing thin cartilage tissue from human mesenchymal stem cells undergoing chondrogenic differentiation. This approach offers a practical platform for producing thin membrane-like tissue constructs for many research and therapeutic applications. Key features • Spheroid-based tissue engineering: Utilizing cell spheroids to build various membrane-like tissues. • Controlled tissue thickness: Frame and mesh constrain thickness and guide lateral fusion of spheroids, enabling uniform and thin tissue for efficient nutrient diffusion. • Scaffold-free construct: After the thin tissue membrane is formed, the frame and mesh can be removed. • Mechanical support: Meshes enable easy handling and can aid in transplantation of the constructs, for example, by allowing them to be wrapped or sutured.

Vestibulo-ocular reflexes (VORs) are compensatory ocular reflexes that maintain stable vision during head movements. In research, VORs encompass angular VOR (aVOR) and off-vertical axis rotation (OVAR) tests, which various groups have employed to assess vestibular function in mice. This protocol outlines the process for measuring VORs in mice, including eye rotation calibration, immobilizing the mouse with a noninvasive setup, configuring the aVOR and OVAR stimulus modes, and interpreting the obtained waveforms to derive VOR values. As technology advances, VORs are expected to yield more qualitative and quantitative insights into the function of the horizontal semicircular canal cristae (HSCC) and the otolith organs. This methodology can serve as a standard for evaluating common vestibular deficits in mice. Key features • The integrated aVOR and OVAR modes enable us to evaluate both the otolith organs and horizontal semicircular canals. • The calibration tools used in our system ensure standardization between different systems, facilitating comparison of results between laboratories. • Animal holders provide a rapid and convenient method for conducting VOR tests without the need for anesthesia or surgery.

In neuropharmacology and drug development, in silico methods have become increasingly vital, particularly for studying receptor-ligand interactions at the molecular level. Membrane proteins such as GABA (A) receptors play a central role in neuronal signaling and are key targets for therapeutic intervention. While experimental techniques like electrophysiology and radioligand binding provide valuable functional data, they often fall short in resolving the structural complexity of membrane proteins and can be time-consuming, costly, and inaccessible in many research settings. This study presents a comprehensive computational workflow for investigating membrane protein-ligand interactions, demonstrated using the GABA (A) receptor α5β2γ2 subtype and mitragynine, an alkaloid from Mitragyna speciosa (Kratom), as a case study. The protocol includes homology modeling of the receptor based on a high-resolution template, followed by structure optimization and validation. Ligand docking is then used to predict binding sites and affinities at known modulatory interfaces. Finally, molecular dynamics (MD) simulations assess the stability and conformational dynamics of receptor-ligand complexes over time. Overall, this workflow offers a robust, reproducible approach for structural analysis of membrane protein-ligand interactions, supporting early-stage drug discovery and mechanistic studies across diverse membrane protein targets. Key features • Applicable to diverse membrane proteins, including ion channels and G protein-coupled receptors (GPCRs), facilitating ligand interactions and dynamic behavior in biologically relevant environments. • Supports the investigation of both natural and synthetic compounds targeting specific receptor subtypes within complex membrane systems. • Combines homology modeling, molecular docking, and molecular dynamics simulations to deliver comprehensive structural and functional insights. • Showcased with the GABA (A) α5β2γ2 receptor subtype and alkaloid from Mitragyna speciosa, with adaptability to a broad range of receptor-ligand systems.

Oxygen tension is a key regulator of early human neurogenesis; however, quantifying intra-tissue O₂ in 3D models for an extended period remains difficult. Existing approaches, such as needle-type fiber microsensors and intensity-based oxygen probes or time-domain lifetime imaging, either perturb the organoids or require high excitation doses that limit the measurement period. Here, we present a step-by-step protocol to measure intra-organoid oxygen in human cerebral organoids (hCOs) using embedded ruthenium-based CPOx microbeads and widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM). The workflow covers dorsal/ventral cerebral organoid patterning, organoid fusion at day 12 with co-embedded CPOx beads, standardized FD-FLIM acquisition (470-nm external modulation, 16 phases at 50 kHz, dual-tap camera), automated bead detection and lifetime extraction in MATLAB, and session-matched Stern-Volmer calibration with Ru(dpp)₃(ClO₄)₂ to convert lifetimes to oxygen concentration. The protocol outputs per-bead oxygen maps and longitudinal patterns stratified by bead location (intra-organoid vs. gel) and sample state (healthy vs. abnormal), enabling direct linkage between developmental growth and oxygen dynamics. Key features • End-to-end workflow linking hCOs generation, on-gel bead embedding, and FD-FLIM oxygen readout. • Longitudinal single-organoid tracking of oxygen tension with bead-level metadata. • Reference-based lifetime calibration and reproducible camera/LED settings. • Ready-to-reuse materials, recipes, timing, and analysis logic.

Insects rely on chemosensory proteins, including gustatory receptors, to detect chemical cues that regulate feeding, mating, and oviposition behaviours. Conventional approaches for studying these proteins are limited by the scarcity of experimentally resolved structures, especially in non-model pest species. Here, we present a reproducible computational protocol for the identification, functional annotation, and structural modelling of insect chemosensory proteins, demonstrated using gustatory receptors from the red palm weevil (Rhynchophorus ferrugineus) as an example. The protocol integrates publicly available sequence data with OmicsBox for functional annotation and ColabFold for high-confidence structure prediction, providing a step-by-step framework that can be applied to genome-derived or transcriptomic datasets. The workflow is designed for broad applicability across insect species and generates structurally reliable protein models suitable for downstream applications such as ligand docking or molecular dynamics simulations. By bridging functional annotation with structural characterisation, this protocol enables reproducible studies of chemosensory proteins in agricultural and ecological contexts and supports the development of novel pest management strategies. Key features • Designed for insect chemosensory research, demonstrated using gustatory receptors from the red palm weevil (Rhynchophorus ferrugineus) as a representative pest species. • Combines OmicsBox for functional annotation with ColabFold for reproducible, high-confidence protein structure prediction in a streamlined workflow. • Accepts input from genome assemblies, transcriptomic datasets, or curated sequence databases, enabling broad application across model and non-model insects. • Produces reliable structural models suitable for downstream studies, including ligand screening, molecular dynamics simulations, and comparative evolutionary analyses.

Following myocardial infarction (MI), myocardial cells undergo cell death, and the necrotic region is replaced by extracellular matrix (ECM) proteins such as collagens. Myofibroblasts are responsible for producing these ECM proteins. Cardiac myofibroblasts are differentiated from resident fibroblasts in response to inflammation. To date, genetically modified mice driven by the Periostin promoter and adeno-associated virus 9 (AAV9) carrying the Periostin promoter have been used for gene transfer into cardiac myofibroblasts. However, these methods require multiple steps and are time-consuming and expensive. Therefore, we developed a method for delivering genes into cardiac myofibroblasts using retroviruses. Specifically, the DNA of the target gene was transfected into Plat-E cells, which are packaging cells, to generate retroviruses. The virus-containing supernatant was then harvested, and the viruses were pelleted by centrifugation and suspended in PBS-containing polybrene. Subsequently, permanent occlusion of the left coronary artery was performed, and 20 μL of viral solution was immediately administered using a 29G needle at a position 1-2 mm below the ligation site in the heart of mice maintained in an open chest state. Using this method, we were able to introduce genes into the myofibroblasts of interest surrounding the MI site. Key features • Retroviruses are taken up only by proliferating cells, enabling highly specific gene transfer into myofibroblasts. • Any gene incorporated into the genome by retroviruses will continue to be expressed over the long term, providing chronic in vivo evaluation. • Myocardial injection targeting the infarct area of the left ventricle shows high infection efficiency in myofibroblasts. • This protocol employs a very small-scale and simple virus concentration method.

The study of whole organs or tissues and their cellular components and structures has been historically limited by their natural opacity, which is caused by the optical heterogeneity of the tissue components that scatter light as it traverses through the tissue, making 3D tissue imaging highly challenging. In recent years, tissue clearing techniques have received widespread attention and undergone rapid development. We recently demonstrated the synthesis of a 2-hydroxyethyl methacrylate (HEMA)-acrylamide (AAm) copolymer. This was achieved using antipyrine (ATP) and 2,2'-thiodiethanol (TDE) as solvents. The resulting solution rapidly embedded tissue samples with a high degree of transparency and is compatible with multiple fluorescence labeling techniques. The method exhibits significant transparency effects across a range of organs, comprising the heart, liver, spleen, lung, kidney, brain (whole and sectioned), esophagus, and small intestine. It can enable volumetric imaging of tissue up to the scale of mouse organs, decrease the duration of the clearing, and preserve emission from fluorescent proteins and dyes. To facilitate the use of this powerful tool, we have provided here a detailed step-by-step protocol that should allow any laboratory to use tissue transparency technology to achieve transparency of tissues and organs. Key features • The method is primarily used for optical tissue clearing. • The method employs a HEMA-AAm copolymer for optical tissue clearing. • This method enables both 2D and 3D fluorescence imaging.

In plants, the apoplast contains a diverse set of proteins that underpin mechanisms for maintaining cell homeostasis, cell wall remodeling, cell signaling, and pathogen defense. Apoplast protein composition is highly regulated, primarily through the control of secretory traffic in response to endogenous and environmental factors. Dynamic changes in apoplast proteome facilitate plant survival in a changing climate. Even so, the apoplast proteome profiles in plants remain poorly characterized due to technological limitations. Recent progress in quantitative proteomics has significantly advanced the resolution of proteomic profiling in mammalian systems and has the potential for application in plant systems. In this protocol, we provide a detailed and efficient protocol for tandem mass tag (TMT)-based quantitative analysis of Arabidopsis thaliana secretory proteome to resolve dynamic changes in leaf apoplast proteome profiles. The protocol employs apoplast flush collection followed by protein cleaning using filter-aided sample preparation (FASP), protein digestion, TMT-labeling of peptides, and mass spectrometry (MS) analysis. Subsequent data analysis for peptide detection and quantification uses Proteome Discoverer software (PD) 3.0. Additionally, we have incorporated in silico-generated spectral libraries using PD 3.0, which enables rapid and efficient analysis of proteomic data. Our optimized protocol offers a robust framework for quantitative secretory proteomic analysis in plants, with potential applications in functional proteomics and the study of trafficking systems that impact plant growth, survival, and health. Key features • Rapid and high-purity collection of Arabidopsis thaliana leaf apoplast flush. • Use of filter-aided sample preparation (FASP) for protein cleaning to obtain high-quality data. • Use of in-house-generated theoretical spectral libraries for efficient and rapid analysis of MS data.

Intestinal glucose absorption has been studied for several decades. However, the different methods available for investigating absorption are often the reason for variability in the results, and it is difficult to measure the relative contribution of paracellular absorption using existing methods. Thus, we have established a new model for measuring glucose absorption. In the isolated in situ vascularly perfused small intestine, the intestinal epithelium is completely preserved, and the entire transport pathway is intact. In the present model, we use radioactive labeled ¹⁴C-d-glucose, which allows for sensitive quantification of glucose absorption even with low luminal concentrations. The described method is optimized for intestinal glucose absorption but can be applied to other macro/micronutrients that can be radioactively labeled. The described procedure is a novel approach for measurements of intestinal nutrient absorption and gut permeability in which luminal nutrient concentrations resemble physiological concentrations. Key features • Sensitive quantification of intestinal glucose absorption at physiologically relevant luminal glucose concentrations. • Accurate distinction between transcellular vs. paracellular glucose absorption using mannitol as an indicator of paracellular intestinal absorption. • Differentiation between apical and basolateral pathways for transport across the intestinal epithelium.

Research on brain disorders, particularly in the field of oncology, requires in vivo models to evaluate various therapeutic approaches, including intracerebral drug delivery. To meet this requirement, the implantation of intracerebral cannulas offers a reliable method for administering candidate therapeutics directly into the brain. This protocol describes a surgical technique for cannula implantation in mice, enabling repeated administration of therapeutic compounds in the context of glioblastoma treatment. The method was designed with an emphasis on using accessible, easy-to-handle, and sterilized tools to optimize surgical outcomes. Particular attention was also given to animal welfare, notably through refined procedures for asepsis, anesthesia, and postoperative care. Key features • This protocol requires specific equipment and surgical mice expertise. • This protocol is dedicated to brain disorders and cancer models, with a particular emphasis on locoregional delivery of drugs and derivatives.