Current protocols最新文献

The aim of this study was to develop an improvised protocol for ex vivo expansion of caprine satellite cells (SCs) using growth factors and their cryopreservation. The SCs were isolated from muscle tissue using collagenase type-I for enzymatic digestion. In vitro culture of SCs was done using Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum (FBS) and 15% horse serum. Immunofluorescence and PCR confirmed positive expression of PAX7 and MyoD, and negative expression of MyHC. Supplementation with 10 ng/ml basic fibroblast growth factor (bFGF) + 100 ng/ml insulin-like growth factor-1 (IGF-1) in the basal culture medium (DMEM + 2% FBS) showed similar results as control (routine culture medium). Subsequently, SCs were successfully cryopreserved using 10% dimethyl sulfoxide and 40% FBS with post-thaw viability of 74.72%. We have successfully established a protocol for the isolation, culture, and cryopreservation of goat SCs. Caprine SCs can be successfully expanded in vitro using growth factors (10 ng/ml bFGF and 100 ng/ml IGF-1) with minimum supplemental FBS in basal culture medium. Additionally, investigations could assess the viability and integrity of cryopreserved goat SCs post-thaw, ensuring that their proliferative capabilities remain intact. © 2025 Wiley Periodicals LLC.

Basic Protocol: Isolation, culture, cost-effective expansion, and cryopreservation of caprine satellite cells

Recombinant monoclonal antibodies (mAbs) are widely used across therapeutic areas, and their glycosylation plays a critical role in product quality, manufacturing consistency, and biosimilarity assessment. A middle-down nuclear magnetic resonance (NMR) method has been developed to profile mAb glycan distribution while preserving the covalent bond between glycan and mAb domains, e.g., the fragment crystallizable (Fc) domain. The workflow uses the immunoglobulin-degrading enzyme from Streptococcus pyogenes (IdeS) to generate Fc fragments that are purified, denatured, and dissolved in urea prior to high-resolution two-dimensional ¹H–¹³C heteronuclear single quantum coherence (2D ¹H–¹³C HSQC) NMR spectrum collection. The resulting anomeric peak distribution reveals major and minor glycan species, including the trimannosyl core, high-mannose variants, and branch-specific galactosylation. Compared with traditional glycan mapping, which requires enzymatic cleavage on glycan and liquid chromatography separation, middle-down NMR provides a non-invasive analysis that preserves glycan integrity and enables comprehensive, semi-quantitative monosaccharide profiling. The method requires 3 to 4 days with ∼4 to 5 hr of hands-on time and can be readily implemented in regulated environments for development and quality control. Basic biochemistry and 2D NMR skills are enough to efficiently apply this protocol in a streamlined workflow. Published 2025. This article is a U.S. Government work and is in the public domain in the USA.

Basic Protocol 1: mAb-Fc sample preparation

Basic Protocol 2: 2D NMR of HSQC peak profile

In situ cryo-electron tomography (cryo-ET) is a rapidly developing approach that enables three-dimensional imaging of cryogenically preserved mammalian cells at up to subnanometer resolution under near-native conditions. Integral to these methods is the successful culture and vitrification of cells directly on electron microscopy (EM) grids. Previous approaches have been individualized for specific cell types. Here, we describe a generalizable protocol applicable to both immortalized and primary mammalian cells. We specifically focus on cryo-ET sample preparation using INS-1E cells, an immortalized rat pancreatic beta-cell line, and primary human fibroblasts. The protocol ensures efficient application and culture of adherent mammalian cells onto EM grids. We then describe the vitrification process by which cultured cells on grids are cryo-preserved in vitreous ice for subsequent cryo-ET imaging. This protocol offers a streamlined approach to mammalian cell-based sample preparation for in situ cryo-ET. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Culture and vitrification of mammalian cells on electron microscopy grids

BossDB is a free and publicly accessible archive for storing and sharing petascale neuroimaging data. Focused on FAIR (i.e., findable, accessible, interoperable, and reusable) principles, it utilizes cloud-based infrastructure and software tools to facilitate data access and analysis. BossDB specializes in storing volumetric electron microscopy (EM) and X-ray microtomography (XRM) imaging data along with associated segmentations, annotations, meshes, and connectomes. Users can browse, access, download, visualize, analyze, and upload data through a variety of interfaces, including the BossDB website, Python software development kit (SDK), and web application programming interface (API). Here we present step-by-step protocols for using these interfaces and BossDB tools to perform each of these tasks. These protocols target any researcher who is interested in learning more about BossDB public datasets, analyzing high-resolution neuroimaging and connectomics data with software tools, or contributing a project to BossDB's catalog of public and private data. © 2025 The Johns Hopkins University Applied Physics Laboratory LLC. Current Protocols published by Wiley Periodicals LLC.

Support Protocol 1: Browsing public data online

Basic Protocol 1: Accessing data with Python

Basic Protocol 2: Accessing data with data API

Basic Protocol 3: Metadata querying via metadata API

Basic Protocol 4: Creating a Neuroglancer visualization

Support Protocol 2: Creating a BossDB account

Basic Protocol 5: Uploading data and metadata

Basic Protocol 6: Uploading a small dataset for private use

Saliva plays a central role in maintaining oral homeostasis by supporting tooth integrity, providing lubrication, and functioning as an antimicrobial wash. It also serves as a transport medium, carrying byproducts and signaling metabolites across oral niches and through the gastrointestinal tract. Because of its biological relevance and ease of collection, saliva is increasingly used as a noninvasive biospecimen for measuring cortisol, cytokines, and metabolites. However, the validity and reliability of saliva as an indicator of local and systemic biomarkers remain under investigation across diverse populations and research applications. Specific patient populations (e.g., individuals with alcohol use disorder) are particularly vulnerable to oral health problems, periodontal disease, and high rates of nicotine use. In addition to behavioral factors (e.g., food, drink, toothbrushing, and mouthwash), patient-specific variables can introduce contaminants such as nicotine and blood into saliva, potentially compromising the accurate measurement of analytes of interest. Protocols that account for possible contaminants are essential to ensure rigorous and reproducible biomarker research. Assessing factors such as pH, flow rate, and visible discoloration helps reduce limitations in analysis and improves interpretation in studies that include heterogeneous populations and health behaviors. Yet, the literature provides limited guidance on standardized methods for saliva collection, processing, and measurement of patient-specific confounders alongside analytes of interest. This protocol addresses these gaps by presenting detailed methodologies for saliva collection and processing, assessment of quantitative and qualitative salivary properties, and quantification of patient-specific modifiers. These approaches support reproducible diagnostics and have applications in populations with high rates of smoking, periodontal disease, and alcohol use. Published 2025. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Saliva collection by the passive drool method

Basic Protocol 2: Processing, storage, and characterization of saliva (visual assessment scale, pH, and flow rate)

Basic Protocol 3: Quantification of cotinine in salivary supernatant

Basic Protocol 4: Quantification of transferrin in salivary supernatant

Chromatin remodeling is a process of regulating gene expression levels by altering chromatin structure, facilitated by chromatin remodeling factors. ISWI (Imitation SWItch), a type of chromatin remodeling factor, plays a crucial role in epigenetic regulatory processes, such as cell differentiation and development. It primarily achieves remodeling effects by promoting the direct sliding of nucleosomes and their relocation on DNA. To study the function and structural characteristics of ISWI, this basic protocol describes a simple and rapid procedure for the large-scale expression and purification of soluble CtISWI_77-1038 and CtATPase_77-722 proteins in Escherichia coli (E. coli). The procedure provides high yields (∼20 mg) and is followed by in vitro measurement of ATP hydrolysis activity. © 2025 Wiley Periodicals LLC.

Basic Protocol: Expression, purification, and ATP hydrolysis activity measurement of CtISWI and CtATPase

The global spread of the arbovirus vector Aedes albopictus has become a major public health concern. In tropical regions, competitive interactions with native species such as Aedes aegypti may have influenced its invasive success. Understanding the bioecological drivers behind its establishment and spread is crucial for informing targeted control measures. Here, we propose experimental settings, based on life history traits, to elucidate the factors that allow Ae. albopictus to colonize new environments, outcompete native species, expand geographically, and ultimately subject invaded regions to risks of arbovirus diseases. To this effect, we designed a series of Basic Protocols listed below. Each protocol simulates environmental conditions in which Ae. albopictus and Ae. aegypti coexist, allowing comparison of their bioecological responses and adaptability to shared resources. The validated results, showing the reproducibility of these methods, provide a foundation for evidence-based strategies to mitigate the public health risks posed by the spread of Ae. albopictus in invaded territories. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Support Protocol: Essential preliminary procedures

Basic Protocol 1: Evaluation of larval habitat occupancy rates

Basic Protocol 2: Evaluation of the female oviposition rate and reproductive potential

Basic Protocol 3: Evaluation of embryonic kinetics

Basic Protocol 4: Evaluation of hatching rate

Basic Protocol 5: Evaluation of longevity

Prostate diseases are increasing globally, necessitating an appropriate model system for investigation. Ex vivo organ models help bridge the gap between organoids, assembloids, and in vivo models. This article represents a robust and reproducible approach for maintaining a functional ex vivo whole-organ prostate gland from the earthworm Eudrilus eugeniae, providing a unique platform for investigating organ-level regeneration, drug screening, and organoid-based research. Sexually mature E. eugeniae worms, which possess a pair of euprostate glands, are pre-processed, meticulously dissected in a sterile environment, and surface sterilized. The glands are treated sequentially in a series of buffers and antibiotic solutions and, without exposure to osmotic stress, are transferred to Leibovitz's L-15 medium containing 10% FBS. The ex vivo prostate glands are incubated at 23°C, simulating the organism's normal temperature. The viability and stress response of the gland are assessed through the rhythmic activity and frequency of prostatic fluids and cells discharged from the anterior duct. Microbial contamination is reduced through early detection and antibiotic treatment. Amputation of the posterior lobe facilitates regeneration via wound closure, contraction, and tissue remodeling, which are enhanced by the use of valproic acid. Released prostatic fluid facilitates noninvasive molecular diagnosis and monitoring of therapeutic response. The cell aggregates released into the medium from the posterior lobe form spheroids, which subsequently grow into organoids and mini-organ structures through self-assembly and cellular bridging. Overall, the developed ex vivo prostate model meets 3R principles for minimizing animal experimentation. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Pre-processing, dissection, and processing of prostate gland for ex vivo maintenance

Support Protocol: Assessing ex vivo viability of prostate gland and handling contamination

Basic Protocol 2: Functional ex vivo prostate gland regeneration, drug screening, and spheroid, organoid, and mini-organ formation

The placenta is a heterogeneous and complex organ with multiple cell types, posing a challenge for the maternal–fetal medicine field to implement single-cell technologies for a deeper characterization of this essential organ. Several protocols use enzymes to digest the tissue and generate single-cell suspensions, but this approach has several shortcomings, including the loss and reduced viability of cells. In this article, we describe a non-enzymatic approach to generate single-cell suspensions from placental tissue with high yield and viability for single-cell RNA sequencing. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of single-cell suspension from freshly collected third trimester human placental sections

Basic Protocol 2: Visualization and determination of cell viability

Alternate Protocol: Counting cells using a hemocytometer

Allosteric transcription factors (aTFs) regulate gene expression in response to small molecules. Engineering aTFs with new ligand specificities expands their utility in environmental monitoring and diagnostics. Here, we present protocols for improving the responsiveness of an aTF, SRTF1, to its non-native ligand, cortisol, based on the SELIS (Seamless Enrichment of Ligand-Inducible Sensors) methodology. First, an aTF library is generated by randomizing the ligand binding pocket. These libraries are then subjected to a selection process to remove protein variants that have lost the ability to repress transcription. The libraries are then plated onto solid media containing the target ligand. The most fluorescent colonies, signifying the most responsive aTFs, are cultured and assayed for the fold change in fluorescence upon induction with the target ligand. Colonies with the highest fold change are cultured, and the plasmid DNA is purified and sequenced. The gene variants are then subcloned into new expression vectors and assayed in a dose-response assay with the target and non-target ligands to have complete characterization of the sensor. Through one round of these protocols, we improved the response of SRTF1 to cortisol by seven-fold; however, the protocols outlined here are applicable to virtually any protein:effector pair. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Library design

Basic Protocol 2: Library construction

Basic Protocol 3: Selection and screening

Basic Protocol 4: Sequencing and validation of hits

Support Protocol 1: Predicting protein:effector structure

Support Protocol 2: Design of mutagenesis primers

Support Protocol 3: Preparation of chemically competent E. coli for single isolate transformations