Current protocols最新文献

{"title":"Redefining Cell Culture Using a 3D Flipwell Co-culture System: A Mimetic for Gut Architecture and Dynamics In Vitro","authors":"Maria A. Beamer,&nbsp;Saori Furuta","doi":"10.1002/cpz1.70107","DOIUrl":"https://doi.org/10.1002/cpz1.70107","url":null,"abstract":"<p>Gut mucosae are composed of stratified layers of microbes, a selectively permeable mucus, an epithelial lining, and connective tissue homing immune cells. Studying cellular and chemical interactions between the gut mucosal components has been limited without a good model system. We have engineered a three-dimensional (3D) multi-cellular co-culture system we coined “3D Flipwell system” using cell culture inserts stacked against each other. This system allows an assessment of the impact of a gut mucosal environmental change on interactions between gut bacteria, epithelia, and immune cells. As such, this system can be utilized in examining the effects of exogenous stimuli, such as dietary nutrients, bacterial infection, and drugs, on the gut mucosa that could predetermine how these stimuli might influence the rest of body. Here, we describe the methods of construction and application of the new 3D Flipwell system we utilized previously in assessing the crosstalk between the gut mucosa and macrophage polarization. We demonstrate the physiological responses of different components of the co-cultures to Sepiapterin (SEP), the precursor of the nitric oxide synthase cofactor tetrahydrobiopterin (BH₄). We reported previously that SEP induces a pro-immunogenic shift of macrophages having acquired an immune suppressive phenotype. We also showed that SEP induces a defense mechanism of commensal gut bacteria. The protocol describing the assembly and use of the 3D Flipwell co-culture system herein would grant its utility in evaluating the concurrent effects of pharmacologic and microbiologic stimuli on gut mucosal components. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: 3D Flipwell construction, assembly, and collagen coating</p><p><b>Basic Protocol 2</b>: Flipwell cell seeding and cell culture</p><p><b>Basic Protocol 3</b>: Addition of bacterial culture to the Flipwell system</p><p><b>Basic Protocol 4</b>: Flipwell disassembly for scanning electron microscopy (SEM) studies</p><p><b>Basic Protocol 5</b>: Immunofluorescence antibody staining for confocal microscopy</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70107","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143431322","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing Reproducibility in Chemotaxis Assays for Caenorhabditis elegans","authors":"Leona Cesar,&nbsp;Julia Morud","doi":"10.1002/cpz1.70106","DOIUrl":"https://doi.org/10.1002/cpz1.70106","url":null,"abstract":"<p>The ability of <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) to navigate complex environments is essential for their survival. This natural behavior is commonly used in chemotaxis assays, which are important tools for studying the function of sensory neurons and neural circuits. Chemotaxis has been essential for discovering fundamental functions in neuronal signaling during the past decades. However, a lack of thoroughly optimized and standardized procedures can lead to variable results that can be difficult to interpret. To improve reproducibility, we optimized several aspects of chemotaxis protocols by testing different odorant concentrations, numbers of worms, and assay durations, as well as the preparation of chemotaxis plates and the washing procedures of worms. The usage of a 2-choice or a 4-choice assay was also evaluated. Our new protocol improves the clarity of results and simplifies worm counting. The protocol optimization is condensed into a 5-day step-by-step protocol that increases the reproducibility of chemotaxis in <i>C. elegans</i>. Compared to previously published chemotaxis protocols, the revised method reduces day-to-day variability using an improved and standardized assay design that ensures clear and reliable results. Several key components in the assay preparation and during the assay have been evaluated based on previous protocols, such as odor concentration, worm density, and assay length. By considering multiple factors that influence the worm's behavior, our optimized protocol enhances the reproducibility of chemotaxis assays in <i>C. elegans</i>, making them more reliable and accessible for studying phenotypes related to olfaction and neural circuit behavior. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Optimized Chemotaxis Assay for <i>C. elegans</i></p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70106","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143431321","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Workflow4Metabolomics (W4M): A User-Friendly Metabolomics Platform for Analysis of Mass Spectrometry and Nuclear Magnetic Resonance Data","authors":"Cédric Delporte,&nbsp;Marie Tremblay-Franco,&nbsp;Yann Guitton,&nbsp;Cécile Canlet,&nbsp;Ralf J. M. Weber,&nbsp;Helge Hecht,&nbsp;Elliott James Price,&nbsp;Jana Klánová,&nbsp;Charlotte Joly,&nbsp;Céline Dalle,&nbsp;Julien Saint-Vanne,&nbsp;Etienne Thévenot,&nbsp;Isabelle Schmitz,&nbsp;Sylvain Chéreau,&nbsp;Sylvain Dechaumet,&nbsp;Binta Diémé,&nbsp;Franck Giacomoni,&nbsp;Gildas Le Corguillé,&nbsp;Mélanie Pétéra,&nbsp;Florence Souard","doi":"10.1002/cpz1.70095","DOIUrl":"10.1002/cpz1.70095","url":null,"abstract":"<p>Various spectrometric methods can be used to conduct metabolomics studies. Nuclear magnetic resonance (NMR) or mass spectrometry (MS) coupled with separation methods, such as liquid or gas chromatography (LC and GC, respectively), are the most commonly used techniques. Once the raw data have been obtained, the real challenge lies in the bioinformatics required to conduct: (i) data processing (including preprocessing, normalization, and quality control); (ii) statistical analysis for comparative studies (such as univariate and multivariate analyses, including PCA or PLS-DA/OPLS-DA); (iii) annotation of the metabolites of interest; and (iv) interpretation of the relationships between key metabolites and the relevant phenotypes or scientific questions to be addressed. Here, we will introduce and detail a stepwise protocol for use of the Workflow4Metabolomics platform (W4M), which provides user-friendly access to workflows for processing of LC–MS, GC–MS, and NMR data. Those modular and extensible workflows are composed of existing standalone components (e.g., XCMS and CAMERA packages) as well as a suite of complementary W4M-implemented modules. This tool suite is accessible worldwide through a web interface and is hosted on UseGalaxy France. The extensible Virtual Research Environment (VRE) provided offers pre-configured workflows for metabolomics communities (platforms, end users, etc.), as well as possibilities for sharing among users. By providing a consistent ecosystem of tools and workflows through Galaxy, W4M makes it possible to process MS and NMR data from hundreds of samples using an ordinary personal computer, after step-by-step workflow optimization. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: W4M account creation, working history preparation, and data upload</p><p><b>Support Protocol 1</b>: How to prepare an NMR zip file</p><p><b>Support Protocol 2</b>: How to convert MS data from proprietary format to open format</p><p><b>Support Protocol 3</b>: How to get help with W4M (IFB forum) and how to report a problem on the GitHub repository</p><p><b>Basic Protocol 2</b>: LC–MS data processing</p><p><b>Alternate Protocol 1</b>: GC–MS data processing</p><p><b>Alternate Protocol 2</b>: NMR data processing</p><p><b>Basic Protocol 3</b>: Statistical analysis</p><p><b>Basic Protocol 4</b>: Annotation of metabolites from LC–MS data</p><p><b>Alternate Protocol 3</b>: Annotation of metabolites from NMR data</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143416477","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Monitoring Mouse Surface Temperature During Stress with a Thermal Camera: A Low-Cost Infrared Videography System for Evaluating Murine Metabolism","authors":"Breanna Page,&nbsp;Carolina Cora,&nbsp;James Reilly,&nbsp;Ryan Reno,&nbsp;Wadak Harbi,&nbsp;Maureen S. Lynes,&nbsp;Michael A. Lynes,&nbsp;Matthew D. Lynes","doi":"10.1002/cpz1.70098","DOIUrl":"https://doi.org/10.1002/cpz1.70098","url":null,"abstract":"<p>Energy is required for life, and organisms obtain their energy from fuel sources to enable both anabolic and catabolic processes. Some of this energy is radiated as heat, which can be quantified as a measure of metabolic rate. In some cases, environmental toxicants can alter metabolic energy in undesirable ways, and characterization of new pharmaceuticals can determine the efficacy of desirable metabolic rate manipulation or identify off-target adverse effects. Current methods to directly measure heat production in laboratory mice are expensive, can be laborious, and make it challenging to monitor animals in ways that are multiplexed, robust, and non-invasive. We present a set of protocols for assembling and deploying a simple, low-cost thermal camera to monitor and record thermogenic activity, modified from prior work. Parts used to build this system currently cost approximately $150 USD and, when assembled, can record mouse temperatures as well as ambient cage temperatures up to twice per second for extended periods. By using multiplexed cameras in a diurnal mouse incubator system, the thermogenic capacity of several mice can be simultaneously recorded and graphed. Exogenous agents and genotypes that alter metabolism can be readily identified with this technology. In this set of protocols, we describe the assembly of the thermal video camera device, its use, and related data capture and analysis methods. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Assembling thermal camera for thermogenic stress test</p><p><b>Basic Protocol 2</b>: <i>In vivo</i> measurement of mouse temperature under different ambient conditions</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70098","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143396822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Efficient Site-Directed Mutagenesis Mediated by Primer Pairs with 3’-Overhangs","authors":"Negar Mousavi,&nbsp;Ethan Zhou,&nbsp;Arezousadat Razavi,&nbsp;Elham Ebrahimi,&nbsp;Paulina Varela-Castillo,&nbsp;Xiang-Jiao Yang","doi":"10.1002/cpz1.70104","DOIUrl":"https://doi.org/10.1002/cpz1.70104","url":null,"abstract":"<p>Site-directed mutagenesis is an essential tool in molecular biology, protein engineering, plasmid engineering and synthetic biology. While the QuickChange method has been one of the most employed methods for site-directed mutagenesis, it is hindered by low efficiency and frequent introduction of unwanted mutations at the primer sites, raising the urgent need for new, more efficient, and reliable methods. Here, we present an optimized site-directed mutagenesis protocol that leverages partially complementary primer pairs with 3’-overhangs to improve mutagenesis efficiency and reduce error rates. Our method significantly enhances success rates, achieving an average efficiency of ∼50% with some instances approaching the ideal threshold of 100%, while also minimizing the time required for mutant generation. Typically, only 3 colonies need to be analyzed per mutagenesis reaction, and a skillful trainee can engineer 1 to 2 dozen mutant plasmids within a week. In addition, with an in-house protocol for preparing highly competent bacterial cells, we have further increased the reliability and cost-effectiveness of the method. Notably, such competent cells have been kept in a liquid nitrogen tank for &gt;12 years with minimal loss of competency. Thus, this refined method offers a robust, efficient, and scalable solution for high-precision gene modification in vitro, with broad applications in protein and plasmid engineering. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: In vitro site-directed mutagenesis using an optimized primer design strategy</p><p><b>Basic Protocol 2</b>: Preparation of high-efficiency chemocompetent DH5α cells for transformation of mutagenized plasmid products</p><p><b>Basic Protocol 3</b>: Transformation of chemocompetent DH5α cells and obtaining bacterial colonies with correctly mutagenized plasmid products</p><p><b>Alternate Protocol</b>: Transformation if glycerol stocks are unavailable</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143404606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Minimal Component, Protein-Free, and Cost-effective Human Pluripotent Stem Cell Cardiomyocyte Differentiation","authors":"Jessika B. Iwanski,&nbsp;Odunayo S. Lawal,&nbsp;William T. Kwon,&nbsp;Isabella Vazquez,&nbsp;Jared M. Churko","doi":"10.1002/cpz1.70099","DOIUrl":"https://doi.org/10.1002/cpz1.70099","url":null,"abstract":"<p>Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become a powerful source for the <i>in vitro</i> modeling of cardiac diseases and various other essential applications, including cardiotoxicity screening and regenerative cell replacement therapies. Although many differentiation protocols have been developed to generate cardiomyocytes from human pluripotent stem cells, these protocols are costly and complex, requiring expensive and often unnecessary components (e.g., B27 medium supplement). In addition, the use of animal-derived growth factors limits their use for regenerative medicine purposes. To address these issues, herein, we have developed an efficient, cost-effective, and protein-free hPSC-CM protocol using only two components: DMEM/F12 basal medium and <span>l</span>-ascorbic acid 2-phosphate. By eliminating xenobiotic and complex components, the efficiency of directed differentiations is increased, the variability between cardiac differentiations is decreased, and the scalability of cell production is enhanced. Adaptation of this efficient, low-cost, and user-friendly cardiac differentiation protocol will enrich the utility and applicability of hPSC-CMs in drug discovery, cell therapies, tissue engineering, disease modeling, precision medicine, and cardiac regenerative medicine. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: hPSC cell culture</p><p><b>Basic Protocol 2</b>: hPSC-CM differentiation</p><p><b>Basic Protocol 3</b>: Characterization of hPSC-CMs by immunofluorescence (IF) imaging</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143388900","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-sensitivity Epimutations Testing with MS-HRM","authors":"Olga Taryma-Leśniak,&nbsp;Katarzyna E. Sokolowska,&nbsp;Magdalena Liput,&nbsp;Tomasz K. Wojdacz","doi":"10.1002/cpz1.70094","DOIUrl":"https://doi.org/10.1002/cpz1.70094","url":null,"abstract":"<p>Epimutation is defined as any heritable change in gene activity that does not affect the actual sequence of DNA. One of the most researched epimutations is promoter methylation of breast cancer susceptibility gene <i>BRCA1</i>. This epimutation may arise de novo in somatic tissues, such as breast or ovarian cancer tissue, but has also been shown to be widely distributed throughout tissues. <i>BRCA1</i> methylation detectable in peripheral blood has been associated with the risk of both breast and ovarian cancer in patients without a germline pathogenic variant status of this gene. The strength of this association suggests that diagnostic screening for the <i>BRCA1</i> epimutation should be considered with potential implications in cancer risk assessment. However, the detection of <i>BRCA1</i> epimutation may be challenging, as it is generally detectable at a very low level in blood. Methylation-sensitive high-resolution melting (MS-HRM) was shown to provide a high sensitivity of methylation detection, and as it is a PCR-based method, epimutation screening based on MS-HRM is labor- and cost-effective. Here we describe the MS-HRM protocol for <i>BRCA1</i> epimutation screening. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: High sensitivity constitutional epimutations testing with MS-HRM</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143380118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Porphyria Diagnostics Part 2: Essential Biochemical Testing for Diagnosis of the Porphyrias","authors":"Vaithamanithi-Mudumbai Sadagopa Ramanujam,&nbsp;Akshata Moghe,&nbsp;Ruksana Huda,&nbsp;Shalonda B. Turner,&nbsp;Karl E. Anderson","doi":"10.1002/cpz1.70092","DOIUrl":"https://doi.org/10.1002/cpz1.70092","url":null,"abstract":"<p>Porphyrins and porphyrin precursors are normally detected in small amounts in healthy individuals but are found in large quantities in the urine, feces, blood, plasma, bone marrow, and liver in patients with various types of porphyrias. These are intermediates, or are derived from intermediates, in the pathway for heme biosynthesis. Heme is synthesized in all body tissues but in the largest amounts in the bone marrow and liver. Accurately measuring these compounds is important for diagnosis and monitoring of porphyrias. In addition, measurement of enzyme activities and mutation analyses by DNA sequencing enables confirmation of a porphyria diagnosis and genetic counseling. Biochemical approaches described here include measurements of porphyrin precursors and porphyrins in the urine, feces, plasma, erythrocytes, and liver, and determination of specific enzyme activities in erythrocytes and other cells. © 2025 Wiley Periodicals LLC.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143380117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Zebrafish Model for Thrombosis and Brain-Behavior Studies","authors":"Jabila Mary,&nbsp;Pudur Jagadeeswaran","doi":"10.1002/cpz1.70096","DOIUrl":"https://doi.org/10.1002/cpz1.70096","url":null,"abstract":"<p>Hemostasis is a defense mechanism for preventing fluid loss in an injured organism with a circulatory system. In mammals, it begins at the site of the injury, with platelet adhesion to components of the subendothelial matrix activating a series of platelet signaling events that ultimately form a primary hemostatic plug. This process is followed by coagulation cascade activation, leading to fibrin formation and reinforcement of the plug. Thrombosis, an intravascular form of fibrin formation, is unpredictable and often associated with severe pain. Despite decades of research, many hemostatic and thrombotic factors remain unknown. To address this, we introduced zebrafish as a genetic model for studying thrombosis and pain. The piggyback knockdown method is used to knock down genes related to hemostasis and other biochemical or physiological pathways. Blood collection in zebrafish is important in characterizing hemostasis mutants such as hemophilia, in assaying blood coagulation, and in counting thrombocytes in diseases such as thrombocytopenia. FACS analysis is used to study thrombocyte development. Thrombocyte aggregation by flow cytometry and a plate tilt method is used in the functional evaluation of thrombocytes in hemostasis. Defects in coagulation can be studied by kinetic blood coagulation assays and bleeding assays. Laser thrombosis is a global assay that measures blood clotting, thrombocyte function, and endothelial function, including stasis. This technique is useful when screening for genes affecting hemostasis in a genome-wide manner. As pain is sensed by the PAR2 receptor, the trypsin aversion assay detects this pathway and can be used to identify genes related to pain pathways. © 2025 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Gene silencing by piggyback knockdown</p><p><b>Basic Protocol 2</b>: Blood collection from adult zebrafish</p><p><b>Basic Protocol 3</b>: Quantification of thrombocytes by FACS analysis</p><p><b>Basic Protocol 4</b>: Thrombocyte aggregation assay by flow cytometry</p><p><b>Alternate Protocol 1</b>: Whole-blood aggregation assay by the plate tilt method</p><p><b>Basic Protocol 5</b>: Kinetic blood coagulation assays</p><p><b>Support Protocol</b>: Preparation of zebrafish thromboplastin</p><p><b>Basic Protocol 6</b>: Laser thrombosis assay</p><p><b>Basic Protocol 7</b>: Chemically induced gill bleeding assay</p><p><b>Alternate Protocol 2</b>: Mechanically induced bleeding assay</p><p><b>Basic Protocol 8</b>: Trypsin aversion assay in zebrafish larvae</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143380097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimizing In Situ Proximity Ligation Assays for Mitochondria, ER, or MERC Markers in Skeletal Muscle Tissue and Cells","authors":"Amber Crabtree,&nbsp;Han Le,&nbsp;Chanel Harris,&nbsp;Ashton Oliver,&nbsp;Andy Barillas,&nbsp;Johnathan Moore,&nbsp;Desiree Ngoc-ha Nguyen,&nbsp;Izabella Marie Rabago,&nbsp;Benjamin Rodriguez,&nbsp;Dominique C. Stephens,&nbsp;Heather K. Beasley,&nbsp;Edgar Garza-Lopez,&nbsp;Kit Neikirk,&nbsp;Margaret Mungai,&nbsp;Larry Vang,&nbsp;Zer Vue,&nbsp;Neng Vue,&nbsp;Andrea G. Marshall,&nbsp;Kyrin Turner,&nbsp;Jianqiang Shao,&nbsp;Sandra Murray,&nbsp;Jennifer A. Gaddy,&nbsp;Celestine Wanjalla,&nbsp;Jamaine Davis,&nbsp;Steven Damo,&nbsp;Antentor O. Hinton Jr","doi":"10.1002/cpz1.70043","DOIUrl":"10.1002/cpz1.70043","url":null,"abstract":"<p>Proximity ligation assays (PLAs) use specific antibodies to detect endogenous protein-protein interactions. PLAs are a highly useful biochemical technique that allow two proteins within proximity to be visualized with fluorescent probes amplified by PCR. While this technique has gained prominence, the use of a PLA in mouse skeletal muscle (SkM) is novel. In this article, we discuss how the PLA method can be used in SkM to study the protein-protein interactions within mitochondria-endoplasmic reticulum contact sites (MERCs). © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Proximity ligation assay for skeletal muscle tissue and myoblast for MERC proteins</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"5 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70043","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143191625","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}