Basic Protocol Research Articles

Highly Effective Chemical Ligation of DNA and l-aTNA.

Chemical ligation of nucleic acids is a significant strategy for the establishment of a variety of functional biological tools and other applications. However, the conventional methodology has been suffering from low reaction efficiency or generation of unnatural structures at the ligation site. This article describes an effective chemical ligation method that connects the hydroxyl group and the monophosphate group at the nick site on the template, generating a natural phosphodiester bond. The method uses only N-cyanoimidazole (CNIm) and divalent metal cations to achieve chemical ligation in a template-directed manner, which can be applied to the ligations of not only DNA but also artificial nucleic acids such as acyclic l-threoninol nucleic acid (l-aTNA). Quantitative ligation is available within 10 min for l-aTNA fragments on an l-aTNA template, and within 2 hr for DNA fragments on a DNA template under optimized conditions. The effective chemical ligation system reported here will enable the development of biotechnology and nanotechnology, including exploration of l-aTNA aptamer via in vitro selection, chemical synthesis of genome-sized DNA and l-aTNA, functionalization of nanostructure, and creation of an l-aTNA-based artificial life in the future. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Chemical ligation using CNIm and divalent metal cation Basic Protocol 2: Imaging analysis of ligation reaction Basic Protocol 3: Mass spectrometry of ligation products Support Protocol: Kinetic analysis.

Highly Adaptable Analysis Tools for Mapping Spatial Features of Cellular Aggregates in Tissues.

Multiplex imaging technologies have developed rapidly over the past decades. The advancement of multiplex imaging has been driven in part by the recognition that the spatial organization of cells can represent important prognostic biomarkers and that simply studying the composition of cells in diseased tissue is often insufficient. There remains a lack of tools that can perform spatial analysis at the level of cellular aggregates (a common histopathological presentation) such as tumors and granulomas, with most analysis packages focusing on smaller regions of interest and potentially missing patterns in the overall lesion structure and cellular distribution. Here, we present protocols to quantitatively describe the cellular structure of entire tissue lesions, built around two novel metrics. The Total Cell Preference Index reports whether a lesion tends to change in density in its central versus peripheral areas and can indicate the extent of necrosis across the entire lesion. The Immune Cell Preference Index then reports whether each immune cell type is located more centrally or peripherally across the entire lesion. The output of both indexes is a single number readout for simple interpretation and visualization, and these indexes can be applied to lesions of any size or shape. This simplifies cross-lesion comparison compared to traditional Euclidian distance-based analysis, which outputs multiple values for each lesion (one for output for each band used in the infiltration analysis). Additionally, this approach can be applied to any slide-scanning multiplexed imaging system, based on either protein or nucleic acid staining. Finally, the approach uses the open-source software QuPath and can be utilized by researchers with a basic understanding of QuPath, with the full analysis able to be applied to pre-generated images within 1 hr. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Image preparation and lesion selection Basic Protocol 2: Total Cell Preference Index and Immune Cell Preference Index.

A Highly Sensitive Tissue-specific qRT-PCR-based Assay for Detection of Melanoma Cells in Tumor-Draining Lymph Nodes.

Melanoma invasion of regional lymph nodes, a critical event in the progression of this disease, is well documented as a poor prognostic factor for patients with melanoma. Assessing lymph node involvement is therefore a routine part of the diagnostic workup for patients presenting with melanoma at a T stage of ≥T2a. In clinical settings, the status and degree of melanoma lymph node involvement is traditionally characterized by histopathological analysis of tissue obtained during a sentinel lymph node biopsy, a labor-intensive and costly approach that requires technically challenging sample preparation and interpretation by a trained pathologist. Alternative approaches that might reduce the financial burden and turnaround time of a sentinel lymph node biopsy workup are therefore desirable. Likewise, the ability to accurately assess lymph node invasion by melanoma in experimental settings is necessary to gain new insights into mechanisms of distant metastasis and tumor-associated immune suppression. With these applications in mind, we recently developed, and describe herein, a tissue-specific qRT-PCR-based protocol for detecting melanoma cells within tumor-draining lymph nodes. Using murine models of lymph-node-invasive and lymph-node-noninvasive melanoma cell lines and a melanin-biosynthesis-pathway-specific Trp2 gene expression assay, we validated this method as a highly sensitive strategy for assessing lymph node involvement by melanoma. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Growth and maintenance of murine melanoma cell cultures Basic Protocol 2: Extraction and quantification of RNA from murine melanoma cultures Basic Protocol 3: cDNA synthesis via reverse transcription Basic Protocol 4: Probe and primer design for TaqMan-based qPCR Basic Protocol 5: qPCR analysis of tissue-specific Trp2 gene expression Basic Protocol 6: In vitro validation of qRT-PCR Trp2 gene expression assay for detection of melanoma cells in murine whole lymph node preparations Basic Protocol 7: In vivo validation of qRT-PCR Trp2 gene expression assay for detection of melanoma cells in murine tumor-draining lymph nodes.

Leveraging the MethMotif Toolkit to Characterize Context-Specific Features and Roles of Methylation Sensitive Transcription Factors.

This article presents a comprehensive guide for using the MethMotif platform, which includes the MethMotif database, the TFregulomeR R package, and a new R library, Forked-TF, designed specifically for analyzing leucine-zipper transcription factors (TFs) that bind DNA as dimers. The MethMotif platform integrates transcription factor binding site (TFBS) motifs with DNA methylation profiles, providing an in-depth analysis of how methylation modulates TF binding across different cell types and conditions. The protocols are organized into three main workflows: (1) Exploration of transcription factor dimerization partners, (2) visualization of methylation-specific TF motifs using TFregulomeR, and (3) characterization of leucine-zipper TF binding patterns with a focus on dimerization. Using the platform's MethMotif database, users can retrieve ChIP-seq and DNA methylation data, intersect TFBS peak regions, and generate TFBS-methylation-informed motif logos. A case study of CEBPB in K562 cells is included to demonstrate the use of the platform, showing how to identify TF dimers, analyze their co-binding behavior, and visualize the impact of DNA methylation on binding specificity. The protocols also provide step-by-step instructions for software installation, data input formats, and interpretation of results, making it accessible to researchers with varying levels of computational expertise. Through these protocols, users can uncover how DNA methylation and TF dimerization influence gene regulatory networks, with a focus on leucine-zipper TFs in a cell-type-specific context. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Exploration of transcription factor dimerization partners Support Protocol 1: Software installation Support Protocol 2: Docker installation Support Protocol 3: Verifying installation Basic Protocol 2: Visualization of alternative cofactors Basic Protocol 3: Characterization of bZIP partners/cofactors Basic Protocol 4: Context-independent and context-dependent analysis.

Label-Free Quantification of Protein Density in Living Cells.

Intracellular water content, W, and protein concentration, P, are essential characteristics of living cells. Healthy cells maintain them within a narrow range, but often become dehydrated under severe stress; moreover, persistent loss of water (an increase in P) can lead to apoptotic death. It is very likely that protein concentration affects cellular metabolism and signaling through macromolecular crowding (MC) effects, to which P is directly related, but much remains unknown in this area. Obviously, in order to study the biological roles and regulation of MC in living cells, one needs a method to measure it. Simple and accurate measurements of P in adherent cells can be based on its relationship to refractive index. The latter can be derived from two or more (depending on the algorithm) mutually defocused brightfield images processed by the transport-of-intensity equation (TIE) that must be complemented by a determination of volume. Here, we describe the experimental considerations for both TIE imaging and for a particular method of cell volume measurement, transmission-through-dye (TTD). We also introduce an ImageJ plugin for solving TIE. TIE and TTD are fully compatible with each other as well as with fluorescence. A similar approach can be applied to subcellular organelles; however, in this case, the volume must be determined differently.© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation for TIE with or without TTD Basic Protocol 2: Acquisition of TIE and TTD images Basic Protocol 3: Calibration of TIE Basic Protocol 4: Measurement of the absorption coefficient of the medium used for TTD Basic Protocol 5: Image processing using Fiji Support Protocol 1: Installation and use of TIE plugin Support Protocol 2: Automation of the double TTD/TIE processing using a Fiji macro.

Isolation and Characterization of Extracellular Vesicles from Natural Killer Cells.

Natural killer (NK) cells are critical components of the immune system, recognized for their ability to identify and eliminate target cells undergoing various forms of stress. Beyond their direct cytotoxic activity, NK cells secrete extracellular vesicles (EVs), which serve as mediators of intercellular communication. These vesicles play an essential role in modulating immune responses and influencing tumoral and post-infection microenvironments. In this study, we present a detailed methodology for the isolation and expansion of human primary NK cells, the accumulation of their EVs, and the subsequent procedures for their isolation and characterization. © 2025 Wiley Periodicals LLC. Basic Protocol 1: NK cell isolation and expansion Basic Protocol 2: NK-EV purification using ultracentrifugation Alternate Protocol: NK-EV isolation using size exclusion chromatography Basic Protocol 3: Characterization of NK-EVs.

Getting Started with Machine Learning for Experimental Biochemists and Other Molecular Scientists.

Machine learning (ML) is rapidly gaining traction in many areas of experimental molecular science for elucidating relationships and patterns in large or complex data sets. Historically, ML was largely the preserve of those with specialized training in fields such as statistics or cheminformatics. Increasingly, however, ML methodologies are becoming part of the standard toolkit for experimental scientists across a range of disciplines. For scientists without a significant background in computer science or statistics, lowering the barrier of entry to these ML techniques is important to broadening access to these powerful methods. Here we provide detailed, step-by-step protocols for performing four ML methods that are particularly useful for applications in biochemistry, cell biology, and drug discovery: hierarchical clustering, principal component analysis (PCA), partial least squares discriminant analysis (PLSDA), and partial least squares regression (PLSR). The protocols are written for the widely used software MATLAB, but no prior experience with MATLAB is required to use them. We include an explanation of each step, pitched at a level to be understood by investigators without any prior experience with ML, MATLAB, or any kind of coding. We also highlight the scientific issues pertaining to selecting and scaling the data to be analyzed. Throughout, we emphasize the relationship between the scientific question and how to choose data and methods that will allow it to be addressed in a meaningful way. Our aim is to provide a basic introduction that will equip experimental chemical biologists, chemists, and other biomedical scientists with the knowledge required to use ML to aid in the design of experiments, the formulation and data-driven testing of hypotheses, and the analysis of experimental data. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Clustering Basic Protocol 2: Principal component analysis Basic Protocol 3: Partial least squares-discriminant analysis Basic Protocol 4: Partial least squares regression.

High-Throughput Culture and DNA Isolation Methods for Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic human fungal pathogen that is widely spread in the environment. The need to screen a large number of environmental or clinical isolates for phenotypic and/or genotypic purposes can be challenging and time consuming when using the protocols that are currently available. We have developed a novel approach that allows one to inoculate cultures, grow individual A. fumigatus isolates, and subsequently isolate DNA of sufficient quality for PCR and/or whole genome sequencing, all in a 96-well format. Compared to currently available methods, our protocols reduce both cost and labor significantly and are compatible with any experimental setup that uses a 96-well format. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: 96-well-format mini slant culture and preparation of spore suspension for Aspergillus fumigatus Basic Protocol 2: PCR-quality DNA isolation Basic Protocol 3: High-purity DNA isolation after mini mycelial mat culture.

Flow Cytometric Bone Marrow Evaluation in Suspected Myelodysplastic Neoplasms.

Myelodysplastic neoplasms (MDS) are acquired heterogeneous clonal hematopoietic stem cell neoplasms, clinically characterized by progressively ineffective hematopoiesis and an increased risk of acute myeloid leukemia. MDS are accompanied by an inflammatory microenvironment and genome instability. Signs of dysplasia can occur in the erythroid, myeloid, monocytic, and megakaryocytic cell lineages and result in anemia, neutropenia, and thrombocytopenia. Multi-parameter flow cytometry can be used to detect aberrant antigen expression patterns typical of MDS, which correlate with cytomorphologically identified dysplasias and provide important information for diagnosis and prognosis. Characteristic findings include an increase in myeloid progenitor cells; aberrant myeloid and erythroid maturation; aberrant marker expression on progenitor cells, granulocytes, and monocytes, which corresponds to lineage infidelity, under-/overexpression, or asynchronous expression; and an increase in monocytes and progenitor cells in chronic myelomonocytic leukemia. The latter represents an independent disease entity with a similar phenotype. In addition, flow cytometry can rule out other causes of cytopenia, such as lymphoma, acute leukemias, paroxysmal nocturnal hemoglobinuria, or systemic mastocytosis with associated hematologic neoplasm. To analyze those features, the European LeukemiaNet recommends a set of markers together with important technical aspects. At least three distinct aberrations in at least two lineages are associated with a high likelihood of MDS. © 2025 Wiley Periodicals LLC. Basic Protocol: Flow cytometric bone marrow evaluation in suspected myelodysplastic neoplasms.

Cryptococcus neoformans Biofilm Formation and Quantification.

Cryptococcus neoformans is an opportunistic fungal pathogen that heads the Fungal Priority Pathogen List published by the World Health Organization (WHO) in 2022. This pathogen is a primary cause of death for immunocompromised individuals (e.g., those with HIV/AIDS, the elderly, immunotherapy recipients), causing approximately 118,000 deaths yearly worldwide. C. neoformans relies on virulence factors that include a polysaccharide capsule, melanin, extracellular enzymes, and thermotolerance to initiate and sustain host infection. Additionally, similar to other fungal pathogens (e.g., Candida albicans), C. neoformans may develop a biofilm organization linked to more persistent cryptococcal infections. Cryptococcal biofilms are highlighted in cases of cryptococcal meningitis, in which biofilm-like structures form that are highly resistant to host immune response and to antifungal therapies. In this regard, fungal biofilm formation has become an important area of study as a means to improve our understanding of the mechanisms regulating biofilm formation and infection and to advance the discovery of antibiofilm therapeutics. To assess biofilm properties and compare across treatments, quantification and evaluation of cell viability are important. Herein, we describe a standardized method to establish a cryptococcal biofilm and quantify total biomass and cell viability. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing and biofilm formation Basic Protocol 2: Biofilm quantification Alternate Protocol: Biofilm viability assay.

Enhancing Visual Discrimination Task: Optimized Mouse Motivation in the Touchscreen Paradigm.

Mouse models are essential for understanding gene function, environmental interaction, and brain structure and function. This is reinforced by the ability of mice to perform complex behavioral tasks. Still, their cognitive assessments often rely on aversive paradigms, such as fear conditioning and the Morris water maze. A promising alternative is the automated touchscreen platform, which enables cognitive tests comparable to those used in humans, such as the Cambridge Neuropsychological Test Automated Battery (CANTAB). This approach enhances standardization and reduces stress by employing appetitive reinforcement. Although widely used in non-human primates, touchscreen testing remains underutilized in rodents despite its potential for cross-species cognitive research. Motivation is key to successful touchscreen tasks, often achieved through water restriction, which mice tolerate well. However, water restriction is a stressful condition, combining negative and positive reinforcement. Here, we propose an alternative that uses citric acid (CA) water to avoid classical food privation in the touchscreen paradigm to mitigate mice's stress. By creating a strong contrast with the reward, we increase the reward's positive valence. We used the touchscreen visual discrimination task to assess the effectiveness of CA water in enhancing motivation. Our results show that administering CA water on training days while allowing access to plain water on weekends reduces the learning phase duration without causing significant weight loss in wild-type C57BL/6J mice. In addition, we observed a strong commitment to performing the pattern dissociation task. This approach offers a welfare-friendly alternative for maintaining motivation in touchscreen-based cognitive tasks while minimizing stress. © 2025 Wiley Periodicals LLC. Basic Protocol: Pattern dissociation paradigm using sour water.

Purification of Low-Complexity Domain Proteins FUS, EWSR1, and Their Fusions.

FET proteins are large multifunctional proteins that have several key roles in biology. The FET family of proteins, including FUS, EWSR1, and TAF15, play critical roles in transcription regulation, RNA processing, and DNA damage repair. These multifunctional RNA- and DNA-binding proteins are ubiquitously expressed and conserved across vertebrate species. They contain low-complexity (LC) domains that allow them to assemble and phase separate but also makes the proteins prone to aggregation. Aberrations in FET proteins, such as point mutations, aggregation, or translocations leading to fusion proteins, have been implicated in several pathologies, including frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), and Ewing sarcoma. In vitro study of FET proteins is hampered by their propensity to aggregate, their disordered structure, and their susceptibility to proteolysis, making high-yield production difficult. Here, we present optimized methods for the purification of full-length FUS, EWSR1, and their fusion proteins. These protocols enable researchers to overcome issues related to aggregation and solubility, facilitating biochemical and biophysical studies of these critical yet complex proteins. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Purification of EWSR1 and FUS proteins Alternate Protocol: Purification for fusion proteins.

Influence of homogenization methods on lichen species detection from environmental DNA metabarcoding

Environmental DNA (eDNA) techniques are increasingly employed in biodiversity monitoring of aquatic and terrestrial animals, plants, and fungi, holding great potential to revolutionize biodiversity assessments. However, sampling and basic laboratory protocols still require refinement to optimize eDNA metabarcoding performance. Homogenization as a pre-treatment for eDNA extraction is known to enhance the concentration and quality of extracted eDNA for some groups of organisms. We previously developed a simple and efficient method for capturing arboreal biodiversity using stemflow as a source of eDNA; however, its performance with or without homogenization had not yet been compared. In this study, we evaluated the performance of two different homogenization methods using eDNA metabarcoding and qPCR assays. Metabarcoding analyses revealed that the method without homogenization detected the fewest species, while nearly identical and higher numbers of species were detected in samples subjected to bead-beating and frozen bead-beating homogenization. Similarly, qPCR analyses revealed that the method without homogenization yielded the lowest DNA concentration, while nearly identical and higher DNA yields were observed for bead-beating and frozen bead-beating homogenization. These findings suggest that, considering cost and effort, the bead-beating method without freezing is the most advantageous.

Robust federated contrastive recommender system against targeted model poisoning attack

Federated recommender systems (FedRecs) have garnered increasing attention recently, thanks to their privacy-preserving benefits. However, the decentralized and open characteristics of current FedRecs present at least two dilemmas. First, the performance of FedRecs is compromised due to highly sparse on-device data for each client. Second, the system’s robustness is undermined by the vulnerability to model poisoning attacks launched by malicious users. In this paper, we introduce a novel contrastive learning framework designed to fully leverage the client’s sparse data through embedding augmentation, referred to as CL4FedRec. Unlike previous contrastive learning approaches in FedRecs that necessitate clients to share their private parameters, our CL4FedRec aligns with the basic FedRec learning protocol, ensuring compatibility with most existing FedRec implementations. We then evaluate the robustness of FedRecs equipped with CL4FedRec by subjecting it to several state-of-the-art model poisoning attacks. Surprisingly, our observations reveal that contrastive learning tends to exacerbate the vulnerability of FedRecs to these attacks. This is attributed to the enhanced embedding uniformity, making the polluted target item embedding easily proximate to popular items. Based on this insight, we propose an enhanced and robust version of CL4FedRec (rCL4FedRec) by introducing a regularizer to maintain the distance among item embeddings with different popularity levels. Extensive experiments conducted on four commonly used recommendation datasets demonstrate that rCL4FedRec significantly enhances both the model’s performance and the robustness of FedRecs.

Developing Monosodium Urate Monohydrate Crystals-Induced Gout Model in Rodents and Rabbits.

Gout is a chronic disease caused by the deposition of monosodium urate monohydrate (MSU) crystals within the body, particularly in one or more joints, which can lead to sudden severe attacks of pain, swelling, redness, and tenderness, known as gout flares. Historically termed the "disease of kings," gout is one of the oldest joint diseases and remains the most common form of inflammatory arthritis haunting humans in the 21st century. It is associated with cardiovascular, metabolic, and renal comorbidities and can lead to reduced mobility and impaired physical function and contributing to work absenteeism. Given its increasing global incidence, novel therapies for gouty arthritis disease are urgently needed. Experimental gout models are indispensable tools for deciphering disease pathogenesis and evaluating the efficacy and side effect of newly developed therapeutics at preclinical stage. Herein, we described a series of highly reproducible acute gout flare and air pouch models in rodents and rabbits that can be used to address various scientific questions relevant to pathological changes and immune responses during and after a gout attack. Animal gout flare models, elicited by MSU crystals, mimic the main histopathological features of human gouty arthritis, including damage to cartilage and joint swelling. Meanwhile, air pouch models serve as a tool to evaluate robust inflammatory cytokine secretion and neutrophil infiltration. This article provides a detailed description of procedures and troubleshooting tips required to reproducibly induce gout flare and air pouch models in animals and critically evaluate the pathogenesis of the disease. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Preparation of monosodium urate crystalline Basic Protocol 2: Development of MSU-induced gout flare model in mice Support Protocol 1: Histological assessment of mouse ankle tissues Basic Protocol 3: Development of MSU-induced gout flare model in rats Basic Protocol 4: Development of MSU-induced gout flare model in rabbits Basic Protocol 5: Development and validation of reference articles in MSU-induced air pouch model in rats Basic Protocol 6: Development and validation of reference articles in MSU-induced air pouch model in mice Support Protocol 2: Flow cytometry of mouse neutrophils in air pouch lavage samples.

Development, Characterization, and Therapeutic Utility of Paclitaxel-Resistant Breast and Gastric Cancer In Vitro and In Vivo Models.

Paclitaxel, one of the most commonly used anticancer agents, is employed in the treatment of a range of malignant tumors. However, resistance is one of the major barriers to successful therapy. Despite its clinical relevance, the molecular mechanisms underlying paclitaxel resistance remain poorly understood. In this protocol, we describe the methods for establishing paclitaxel-resistant tumor models both in vitro and in vivo, and how we investigated the underlying mechanisms of resistance. Additionally, we evaluated the potential of combination therapies to overcome paclitaxel resistance in these models. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Generation of paclitaxel-resistant breast cancer model in vivo. Basic Protocol 2: Generation of paclitaxel-resistant gastric cancer model in vitro. Basic Protocol 3: Validation of drug resistance in vivo. Basic Protocol 4: In vitro and in vivo evaluation of combination therapy in paclitaxel-resistant models.

Preparation of Boranophosphate DNA via the Boranophosphotriester Method and Synthesis of P-modified DNAs from Boranophosphate DNA via an Acyl Phosphite Intermediate.

Boranophosphate DNAs, in which one of the nonbridging oxygen atoms in the phosphate moiety is replaced with a borano group, are promising antisense oligonucleotide candidates because of their high nuclease resistance and low cytotoxicity. In Basic Protocol 1, we describe the synthesis of a dithymidylate boranophosphodiester via a boranophosphotriester method. Using the boranophosphotriester method, the condensation of a deoxyribonucleoside 3'-O-boranophosphodiester, which contains a borano group with a 5'-hydroxy group of another deoxyribonucleoside, followed by the deprotection of a protecting group at the phosphorus center, is conducted to yield a boranophosphodiester. A boranophosphodiester is also a useful synthetic precursor, and several P-modified nucleic acid derivatives can be successfully synthesized via the conversion reaction of a boranophosphodiester. In Basic Protocol 2, we describe the conversion reaction of the boranophosphodiester through an acyl phosphite intermediate. During the conversion reaction, various P-modified nucleic acid derivatives, such as a phosphorothioate diester, phosphotriester, phosphoramidate, phosphorothioate triester, and phosphorothioamidate, are obtained in good yields (69% to 93%). © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of dithymidylate boranophosphodiester Support Protocol 1: Preparation of 3-[(trimethylsilyl)oxy]propanenitrile Support Protocol 2: Preparation of PyNTP Support Protocol 3: Preparation of compound 7 Basic Protocol 2: One-pot synthesis of P-modified nucleic acid derivatives.

Extracting Protoplasts from Filamentous Fungi Using Extralyse, An Enzyme Used in the Wine Industry.

The ability to extract protoplasts has contributed significantly to the study of fungi and plants. Protoplasts have historically been used to determine chromosome number via pulsed-field electrophoresis and for the functional characterization of genes via protoplast transformation. More recently, protoplasts have been used to extract the high-molecular-weight DNA required for long-read sequencing projects. The availability of efficient protoplast extraction protocols is thus integral to the study and experimental manipulation of model and non-model fungi. One major hurdle to the development of such protocols has been the discontinuation of enzymes and enzyme cocktails used to digest the fungal cell wall. Here, we provide five protoplast extraction protocols for use in various filamentous ascomycete species spanning the genera Ceratocystis, Fusarium, Metarhizium, Ophiostoma, and Sclerotinia. These protocols all use an inexpensive, readily available enzyme cocktail called Extralyse, a commercially available product commonly used in the wine making industry. Using this enzyme cocktail overcomes reliance on the laboratory-grade enzymes that have frequently been discontinued and are often cost prohibitive at the concentrations required. The protocols described here will allow further research, including genome editing, to be conducted in these fungal genera. Importantly, these protocols also provide a starting point for the development of protoplast extraction techniques in other filamentous fungi. This resource can therefore be used to expand the molecular toolkits available for fungi beyond the species described here, including those with relevance in both medical and biotechnological industries. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Protoplast extractions from Ceratocystis eucalypticola and Ceratocystis fimbriata Basic Protocol 2: Protoplast extractions from Fusarium circinatum Basic Protocol 3: Protoplast extractions from Metarhizium acridum, Metarhizium brunneum, and Metarhizium guizhouense Basic Protocol 4: Protoplast extractions from Ophiostoma novo-ulmi Basic Protocol 5: Protoplast extractions from Sclerotinia sclerotiorum.

Imaging the Intestinal Transcriptome With Multiplexed Error-Robust Fluorescence In Situ Hybridization (MERFISH).

Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is a massively multiplexed single RNA-molecule imaging technique capable of spatially resolved single-cell transcriptomic profiling of thousands of genes in millions of cells within intact tissue slices. Initially introduced for brain tissues, MERFISH has since been extended to other tissues, where rapid RNA degradation during the preparation process can pose challenges. This protocol outlines the application of MERFISH in one such challenging tissue, the mammalian gastrointestinal tract. We describe two complementary protocols leveraging either fresh frozen or fixed frozen approaches and describe methods for combining RNA imaging with immunofluorescence. While these protocols were designed and validated in gut tissues, we anticipate that they will be useful resources for the application to other challenging tissue types. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Fixed-frozen sample preparation Basic Protocol 2: Fresh-frozen sample preparation Basic Protocol 3: Encoding probe construction Basic Protocol 4: MERFISH imaging Basic Protocol 5: Image decoding Support Protocol 1: Coverslip silanization Support Protocol 2: Poly-d-lysine (PDL) coating of the coverslips Support Protocol 3: Hybridization buffer preparation Support Protocol 4: Trolox quinone stock preparation Support Protocol 5: TCEP stock preparation Alternate Protocol 1: MERFISH-compatible immunofluorescent boundary stains in fresh frozen tissue Alternate Protocol 2: Immunofluorescent boundary stains with methacrylate-NHS-anchored antibodies for PFA-fixed samples Alternate Protocol 3: Guanidine-HCl tissue clearing.

Optimized, Efficient Measurement of the Expression of Undifferentiated Stem Cell Markers in Human Induced Pluripotent Stem Cells (iPSCs) by Flow Cytometry.

Induced pluripotent stem cells(iPSCs) have revolutionized the fields of regenerative medicine, disease modeling, and drug discovery. However, the usage of iPSCs for various applications has been hampered by the observed line-to-line variability in their differentiation capacity. Therefore, it is important to verify the pluripotent status of iPSCs. A very effective way to define the pluripotent state of iPSCs is by evaluating the expression of established undifferentiated stem cell markers. A bona fide iPSC must have high, homogeneous expression of these markers. Here, we present a cost-effective platform that can be readily utilized by researchers to define the pluripotency status of iPSCs by measuring the expression of surface and intracellular markers by flow cytometry. © 2025 Wiley Periodicals LLC. Basic Protocol 1: iPSC culture and collection for flow cytometry analysis Basic Protocol 2: Staining of iPSCs for extracellular and intracellular undifferentiated stem cell markers Basic Protocol 3: Flow cytometry acquisition Basic Protocol 4: Flow cytometry data analysis.