Bio-protocol最新文献

Intravenous hemostats have shown significant promise in prolonging survival for severe noncompressible and internal injuries in preclinical animal models. Existing approaches include the use of liposomes with or without procoagulant enzymes, as well as polymer nanoparticles or soluble biopolymers. While these methods predominantly target or mimic tissue components that are present during coagulation, such as activated platelets and collagen, they may not account for the loss of fibrinogen, which is not only key to clot formation but also the first protein to fall below critical levels in dilutional coagulopathy. This protocol describes the synthesis and in vitro or ex vivo characterization of a crosslinkable nanoparticle system that seeks to address dilutional coagulopathy by leveraging the critical gelation concentration and bioorthogonal click chemistry. The system was shown to only gel at high nanoparticle and crosslinker concentrations, increase the rate of platelet recruitment, and decrease the rate of clot degradation in a low-fibrinogen environment, providing a platform for treating severe hemorrhage in a coagulopathic environment. Ultimately, the contents of this protocol may assist researchers in the in vitro characterization and screening of other crosslinkable nanoparticle systems or hemostats, with potential expansions to other categories of coagulation dysfunction, such as embolism treatment. Key features • A protocol for the synthesis of nanoparticles with activated-platelet-binding moieties to mimic fibrin. • In vitro and ex vivo assays assessing complement activation, accumulated platelet recruitment, platelet recruitment under hemodilution, coagulation potential, and clot lysis. • The inclusion of hemodiluted and plasminolytic conditions creates a more physiologically relevant environment for screening of hemostatic agents. • The use of a two-component system helps reduce complement activation in intravenous therapies.

The tissue explant culture (histoculture) is a method that involves maintaining small pieces taken from an organ ex vivo or post mortem in a controlled laboratory setting. Such a technique has a number of advantages: unlike the 2D, organoid, or on-chip cultures, tissue explants preserve the whole complexity of the original tissue in vivo, its structure, extracellular matrix, and the diverse cell populations, including resident immune cells. The explant culture method can be applied to human tissue specimens obtained from biopsies or autopsies, provided that proper ethical protocols are followed. This avoids the difficulties that may arise in translating results obtained on animal models into biomedical research for humans. This advantage makes histocultures especially desirable for studying human pathogenesis in the course of infectious diseases. The disadvantage of the method is the limited lifespan of the cultured tissues; however, a number of approaches allow extending tissue viability to a period sufficient for observing the infection onset and development. Here, we provide a protocol for lung explant maintenance that allows tracing the local effects of infection with SARS-CoV-2 in humans. Further applications of the lung tissues cultured according to this protocol include, but are not limited to, histochemical and immunohistochemical studies and microscopy, FACS, qPCR, and ELISA-based analysis of the conditioned culture media. Key features • The protocol relies on lung tissue culture on collagen rafts at the air-liquid interface, followed by infection with viral agents. • The developed system provides a laboratory-controlled model to investigate the mechanisms of SARS-CoV-2 infection and allows further histological/immunohistochemical, qPCR, FACS, and xMAP cytokine analysis. • Successful establishment of explant culture requires basic cell culture experience. Successful viral infection requires access to a BSL3 laboratory and relevantly trained personnel.

Primary cilia are evolutionarily conserved organelles that play critical roles in brain development. In the developing cortex, neural progenitors extend their primary cilia into the ventricular surface, where the cilia act as key signaling hubs. However, visualizing these cilia in a systematic and intact manner has been challenging. The commonly used cryostat sectioning only provides a limited snapshot of cilia on individual sections, and this process often disrupts the ciliary morphology. By contrast, the previously established whole-mount technique has been shown to preserve ciliary architecture in the adult mouse cortex. Here, we adapt and optimize the whole-mount approach for embryonic and neonatal brain, allowing robust visualization of ciliary morphology at the ventricular surface during development. This protocol describes step-by-step procedures for whole-mounting and immunostaining delicate embryonic and neonatal mouse cortices, enabling direct visualization of cilia in neural progenitors in the developing brain. Key features • This protocol adapts the whole-mount technique and applies it to delicate embryonic samples from embryonic day 12 (E12) to neonatal brain (P3). • This protocol details the necessary steps to achieve intact and direct visualization of cilia in the developing mouse cortex. • This protocol also provides the necessary steps for the dissection and visualization of cilia on the lateral ganglionic eminences (LGE) and medial ganglionic eminences (MGE).

The cellular secretome is a rich source of biomarkers and extracellular signaling molecules, but proteomic profiling remains challenging, especially when processing culture volumes greater than 5 mL. Low protein abundance, high serum contamination, and sample loss during preparation limit reproducibility and sensitivity in mass spectrometry-based workflows. Here, we present an optimized and scalable protocol that integrates (i) 50 kDa molecular weight cutoff ultrafiltration, (ii) spin column depletion of abundant serum proteins, and (iii) acetone/TCA precipitation for protein recovery. This workflow enables balanced recovery of both low- and high-molecular-weight proteins while reducing background from serum albumin, thereby improving sensitivity, reproducibility, and dynamic range for LC-MS/MS analysis. Validated in human mesenchymal stromal cell cultures, the protocol is broadly applicable across diverse cell types and experimental designs, making it well-suited for biomarker discovery and extracellular proteomics. Key features • Enables efficient concentration and cleanup of ≥5-500 mL of conditioned media, suitable for low-abundance secreted protein recovery. • Combines 50 kDa ultrafiltration, optional HSA/IgG depletion, and acetone/TCA precipitation for robust removal of serum contaminants and improved signal-to-noise. • Adaptable to various mammalian cell types and serum-free or serum-containing media; scalable for adherent and suspension cultures.

The exploration of microbial genomes through next-generation sequencing (NGS) and genome mining has transformed the discovery of natural products, revealing an immense reservoir of previously untapped chemical diversity. Bacteria remain a prolific source of specialized metabolites with potential applications in medicine and biotechnology. Here, we present a protocol to access novel biosynthetic gene clusters (BGCs) that encode natural products from soil bacteria. The protocol uses a combination of Oxford Nanopore Technology (ONT) sequencing, de novo genome assembly, antiSMASH for BGC identification, and transformation-associated recombination (TAR) for cloning the BGCs. We used this protocol to allow the detection of large BGCs at a relatively fast and low-cost DNA sequencing. The protocol can be applied to diverse bacteria, provided that sufficient high-molecular-weight DNA can be obtained for long-read sequencing. Moreover, this protocol enables subsequent cloning of uncharacterized BGCs into a genome engineering-ready vector, illustrating the capabilities of this powerful and cost-effective strategy. Key features • This protocol enables bioprospection through cloning of a novel BGC identified in an ONT bacterial draft genome. • A combination of ONT sequencing, antiSMASH, and TAR cloning can be used to clone BGCs from bacteria into a vector. • Cost-effective strategy for the discovery of BGCs of diverse natural product classes, including nonribosomal peptides, polyketides, and RiPPs. • Overnight sequencing in-house using cheap and easy-to-use instruments such as MinION, which allows multiplexing.

Expansion microscopy (ExM) enables nanoscale imaging of biological structures using standard fluorescence microscopes. Accurate labeling of cytoskeletal filaments, such as microtubules, remains challenging due to structural distortion and labeling inaccuracy during sample preparation. This protocol describes an optimized method combining detergent extraction and NHS-ester labeling for high-precision visualization of microtubules in expanded samples. Cytoplasmic components and membranes are selectively removed, preserving the ultrastructure of the microtubule network. Microtubules are digested into peptides during expansion and subsequently labeled at their N-termini using NHS-ester dyes, eliminating the need for antibodies. Effective fluorophore displacement of ~1 nm or lower is achieved, depending on the applied expansion factor. The protocol is compatible with both in vitro and cellular samples and can be integrated into a wide range of ExM workflows. Labeled microtubules can serve as internal reference standards for correcting expansion factors in ExM datasets. Key features • Employs detergent extraction with accessible commercial reagents to isolate cytoskeletal structures and reduce background from membranes and cytoplasmic proteins in fluorescence microscopy. • Avoiding excessive aldehyde fixation preserves amines required for gel polymer integration while NHS-ester labeling of tubulin amines reduces linkage error, enabling accurate molecular localization. • Compatible with post-expansion workflows; labels newly generated peptide N-termini after digestion, enabling high-resolution fluorescence imaging with minimal linkage error and high signal-to-noise ratio (SNR). • Suitable for integration into diverse ExM protocols and useful as a reference standard for expansion factor correction.

The pancreatic islet, the only type of tissue that secretes insulin in response to elevated blood glucose, plays a vital role in diabetes development and treatment. While various islet vascularization strategies have been developed, they have been hindered by major limitations such as relying on pre-patterning and the inability to span long distances. Furthermore, few strategies have demonstrated robust enough vascularization in vivo to support therapeutic subcutaneous islet transplantation. Using adaptive endothelial cells (ECs) reprogrammed by transient expression of the ETS Variant Transcription Factor 2 (ETV-2) gene, we have physiologically vascularized human islets within a generic microchamber and have achieved functional engraftment of human islets in the subcutaneous space of mice. Such adaptive ECs, which we term reprogrammed vascular ECs (R-VECs), have been proven to be a suitable tool for both in vitro disease modeling and in vivo transplantation of not only islets but also other organoids. Key features • This protocol contains two parts: the in vitro and in vivo parts, both utilizing adaptable endothelial cells to functionally vascularize human islets. • The in vitro portion of this protocol describes the method to culture human islets in a vascular bed within a large and commercially available microchamber. • The in vivo portion of this protocol provides a step-by-step procedure to reverse hyperglycemia in streptozotocin-induced diabetic mice.

Peripheral nerve injuries (PNIs) often result in incomplete functional recovery due to insufficient or misdirected axonal regeneration. Balanced regeneration of myelinated A-fibers and unmyelinated C-fibers is essential for functional recovery, making it crucial to understand their differential regeneration patterns to improve PNI treatment outcomes. However, immunochemical staining does not clearly differentiate between A- and C-fiber axons in whole-mount nerve preparations. To overcome this limitation, we developed a modified protocol by optimizing the immunostaining to restrict the antibody access to myelinated axons. This enables visualization of A-fibers by myelin sheath labeling, while allowing selective staining of unmyelinated C-fiber axons. As a result, A- and C-fibers can be reliably distinguished, facilitating accurate analysis of their regeneration in both normal and post-injury conditions. Combined with confocal microscopy, this approach supports efficient screening of whole-mount nerve preparations to evaluate fiber density, spatial distribution, axonal sprouting, and morphological characteristics. The refined technique provides a robust tool for advancing PNI research and may contribute to the development of more effective therapeutic strategies for nerve repair. Key features • Visual separation of myelinated A-fibers and unmyelinated C-fibers is achieved by restricting the penetration of axon-labeling antibodies through the myelin sheaths. • The protocol also distinguishes A- and C-fibers based on the types of associated Schwann cells. • The protocol is specially designed to distinguish between A- and C-fibers as well as their morphological features in whole-mount nerve preparations. • The protocol does not require specialized reagents, equipment, or techniques, making it highly accessible and reproducible across different research settings.

Characterizing the morphology of amyloid proteins is an integral part of studying neurodegenerative diseases. Such morphological characterization can be performed using atomic force microscopy (AFM), which provides high-resolution images of the amyloid protein fibrils. AFM is widely employed for visualizing mechanical and physical properties of amyloid fibrils, not only from a biological and medical perspective but also in relation to their nanotechnological applications. A crucial step in AFM imaging is coating the protein of interest onto a substrate such as mica. However, existing protocols for this process vary considerably. The conventional sample preparation method often introduces artifacts, particularly due to deposition of excess salt. Hence, an optimized protocol is essential to minimize salt aggregation on the mica surface. Here, we present an optimized protocol for coating amyloid proteins onto mica using the dip-washing method to eliminate background noise. This approach improves the adherence of protein to the mica surface while effectively removing residual salts. Key features • The protocol introduces a new method to coat protein samples onto mica sheets for AFM imaging. • It presents a dip-washing technique aimed at removing excess salt deposited on the mica sheet, thereby minimizing imaging artifacts. • This protocol can be used for analyzing amyloid fibrillation mechanisms as well as capturing time-dependent fibrillation dynamics under various physiological conditions. • It also provides clear stepwise washing instructions that balance the salt removal and retention of protein fibrils on the mica.

Genome walking is a classical molecular biology technique used to amplify unknown regions flanking known DNA sequences. Genome walking holds a vital position in the areas associated with molecular biology. However, existing genome-walking protocols still face issues in experimental operation or methodological specificity. Here, we propose a novel genome-walking protocol based on bridging PCR. The critical factor of this protocol is the use of a bridging primer, which is made by attaching an oligomer (or tail primer sequence) to the 5' end of the walker primer 5' region. When the bridging primer anneals to the walker primer site, this site will elongate along the tail of the bridging primer. The non-target product (the main contributor to background in genome walking), defined by the walker primer, is lengthened at both ends. In the next PCR(s), the annealing between the two lengthened ends is easier than the annealing between them and the shorter tail primer. As a result, this non-target product is eliminated without affecting target amplification. Key features • This bridging PCR protocol, built upon the technique developed by Lin et al. [1], is universal. • The bridging primer is made by attaching a tail DNA to the 5' end of the walker primer 5' region. • Lengthening of non-target DNA by both ends of bridging primer results in intrastrand annealing or hairpin formation, the basis for the removal of non-target background.