Current protocols最新文献

Aptamer–drug conjugates (ApDCs) represent a powerful platform for targeted drug delivery, offering the potential to enhance therapeutic efficacy while minimizing systemic toxicity. However, conventional methods for ApDC preparation often involve complex, multistep procedures with limited control over drug loading and site specificity. This protocol describes a modular and automated strategy for the synthesis and structural optimization of ApDCs using custom-designed phosphoramidites that incorporate anticancer drugs and functional moieties. The customized phosphoramidite is compatible with standard solid-phase oligonucleotide synthesis, enabling the automated and efficient construction of programmable aptamer–drug conjugates (PApDCs) for targeted drug delivery. We also highlight the programmable optimization of aptamer structures to meet clinical needs. The protocol offers a streamlined and versatile platform for the construction of functional ApDCs, facilitating their application in receptor-targeted chemotherapy and nucleic acid–based therapeutics. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis and purification of phosphoramidites

Basic Protocol 2: Automated solid-phase synthesis and characterization of ApDCs

Unattended endotoxin (ETX) contamination in biological samples constitute a major challenge for in vitro and in vivo applications. Besides being potentially life-threatening, ETX contamination is especially relevant in global transcriptome analyses, where competing ETX stimulation can significantly skew the final gene expression profile. Our studies in mice and cultured skin epithelial cells (epidermal keratinocytes) aiming to characterize the effect of antibodies such as AK23 immunoglobulins (IgG directed against the cell-cell adhesion molecule desmoglein [DSG] 3) in the autoimmune disease pemphigus vulgaris (PV) revealed that laboratory-produced and even commercial control antibodies can exhibit non-negligible ETX contaminations. Moreover, these contaminants are extremely difficult to remove. To overcome these challenges, we have devised a simple yet nontoxic and scalable two-step protocol to efficiently reduce ETX levels during or after the IgG purification process. It consists firstly of 0.5 M NaOH pre-treatment of all devices, including the protein A resin, used during IgG sanitization and purification, in parallel with meticulous in-process monitoring of ETX levels. Secondly, before IgG elution from protein A, ETX is stripped from IgG by ion-exchange with the common amino acid arginine. This two-step approach successfully reduces ETX by >95% from hybridoma-derived, laboratory-produced AK23 IgG, as well as patient PV and control IgG, resulting in an 85% IgG recovery rate and ETX levels compatible with U.S. Pharmacopeia guidelines. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Sanitization of devices and protein A resin with 0.5 M NaOH

Basic Protocol 2: On-column stripping of ETX from AK23 IgG

Basic Protocol 3: Quality control of sanitized AK23 IgG

High-throughput screening (HTS) of proteins is used in a wide range of applications across the biology, biotechnology, and medicine disciplines. These include yield optimization, drug or biomarker discovery, and protein engineering, among others. Factors that need to be considered in designing high-throughput protein expression and screening methods (be that for expression, activity, stability, or binding assays), include the required yield, reproducibility, solubility, stability, purity, and activity of the protein. Thus, larger culture volumes and time-consuming manual protein extraction and purification steps are normally required to produce enough protein of appropriate purity. This limits the type of assay and number of protein variants that can be simultaneously tested in an experiment. Here, we describe a HTS protocol that allows the overnight expression, export, and assay of recombinant proteins from Escherichia coli cells in the same microplate well. The protocol uses a recently described Vesicle Nucleating peptide (VNp) technology that promotes high yield vesicular export of functional proteins from E. coli into the culture medium. The resulting protein is of sufficient purity and yield that it can be used directly in plate-based enzymatic assays without additional purification. This simple single-plate protocol allows itself to a wide range of high-throughput research and development screening applications, ranging from streamlining protein production and identification of activity enhancing mutations, to ligand screening for basic research, biotechnological and drug discovery applications. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Expression, export, and isolation of vesicular-packaged recombinant protein

Support Protocol 1: 96-well plate cold-shock transformation

Support Protocol 2: In-plate affinity-tag protein purification

Support Protocol 3: Example in-plate enzymatic assay

Microscopic examination of tissue morphology using hematoxylin and eosin (H&E) staining is a standard practice in histopathology for analyzing tissue specimens. In preclinical models of cancer, H&E-stained tissue sections are routinely used to assess tumor development and progression, including parameters such as tumor number, size, and histopathological features. Recent advances in whole-slide scanning and digital pathology now enable quantitative analysis of high-resolution digital images on a computer, replacing the need to examine physical tissue slides under a microscope. However, quantifying tumors on H&E-stained digital sections remains challenging, highlighting the need for accessible and reproducible methods that can be adapted to diverse tumor models. Here, we present two efficient workflows using QuPath, an open-source software for digital pathology and whole-slide image analysis, to quantify tumor multiplicity (i.e., the number of tumors) and tumor burden (e.g., tumor surface area) based on digitally captured H&E-stained tissue sections. As a proof of concept, we apply these workflows to two distinct lung cancer models: (1) an orthotopic model in which Lewis lung carcinoma (LLC) cells are delivered intranasally or intravenously into syngeneic C57BL/6 mice, resulting in well-separated, easily distinguishable tumors amenable to manual quantification; and (2) a genetically engineered mouse model (GEMM) with lung-specific expression of oncogenic Kras^G12D, characterized by infiltrative tumors that are less clearly demarcated due to integration with stromal and immune components, making them more suitable for semi-automated analysis. These Basic Protocols provide accessible and reproducible methods for quantifying tumor development that can be adapted to diverse cancer models. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Quantification of tumor multiplicity and tumor burden using a manual method

Basic Protocol 2: Quantification of tumor burden using a semi-automated method

Skeletal muscle fiber type composition affects muscle function, metabolism, and disease vulnerability. In addition, muscle fiber type analysis informs disease diagnosis and underlying pathophysiology. Multiple methodologies can be used to assess muscle fiber type; however, immunofluorescence (IF) for myosin heavy chain (MyHC) isoforms is the most widely used modern approach due to its relative ease, time-effectiveness, single-cell resolution, and capacity to preserve spatial positioning within the native tissue architecture. Here, we present a protocol for IF for MyHC labeling on formalin-fixed paraffin-embedded (FFPE) mouse and human muscle sections. We then describe a modified procedure for fiber type analysis of the intact mouse lower hindlimb, enabling high-throughput muscle composition and morphological analysis across distinct muscles on a single tissue section. Traditionally, IF labeling for MyHC isoforms required fresh tissue flash-frozen in liquid nitrogen–cooled isopentane, which, while effective, presents challenges for sample processing and preservation, long-term storage, transport, and biosafety. Comparatively, embedding tissue in paraffin after formalin fixation streamlines clinical workflows, preserves morphology, improves long-term sample stability, and simplifies sample storage and transport. Furthermore, FFPE effectively inactivates most infectious agents, which can be retained in frozen sections. Thus, FFPE samples are typically safe for standard laboratory handling and are not classified as biohazardous. This approach can be adapted for use with a range of downstream applications, including integration of fiber type analysis with emerging next-generation techniques that favor FFPE samples. In sum, this method offers a robust alternative to traditional fresh-frozen protocols and allows for simultaneous fiber type analysis across multiple muscle tissues. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Multiplex Immunofluorescence for MyHC Labeling in FFPE Skeletal Muscle Tissues

Alternate Protocol 1: Immunofluorescence for MyHC 2x Labeling in FFPE Skeletal Muscle Tissues

Alternate Protocol 2: Multiplex Immunofluorescence for MyHC Labeling in FFPE Whole Hindlimb Sections

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