Bio-protocol最新文献

{"title":"Isolation and Co-culture of Paneth Cells and Intestinal Stem Cells.","authors":"Ryosuke Isotani, Masaki Igarashi, Masaomi Miura, Toshimasa Yamauchi","doi":"10.21769/BioProtoc.5449","DOIUrl":"10.21769/BioProtoc.5449","url":null,"abstract":"<p><p>Crypts at the base of intestinal villi contain intestinal stem cells (ISCs) and Paneth cells, the latter of which work as niche cells for ISCs. When isolated and cultured in the presence of specific growth factors, crypts give rise to self-renewing 3D structures called organoids that are highly similar to the crypt-villus structure of the small intestine. However, the organoid culture from whole crypts does not allow investigators to determine the contribution of their individual components, namely ISCs and Paneth cells, to organoid formation efficiency. Here, we describe the method to isolate Paneth cells and ISCs by flow cytometry and co-culture them to form organoids. This approach allows the determination of the contribution of Paneth cells or ISCs to organoid formation and provides a novel tool to analyze the function of Paneth cells, the main component of the intestinal stem cell niche. Key features • This protocol introduces the method for isolating Paneth cells and <i>Lgr5+</i> ISCs by flow cytometry and co-culturing them. • This protocol allows analyzing the effect of genetic or biochemical modifications of Paneth or <i>Lgr5⁺</i> ISCs on organoid formation. • This protocol provides a new platform to analyze Paneth cell function.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5449"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457840/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152393","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":"A Method to Inoculate Millet Grain-Colonized <i>Fusarium pseudograminearum</i> on Wheat to Obtain Reproducible Disease Symptoms.","authors":"Zhe Tang, Weiran Kong, Ming Xu, Xin Zhang","doi":"10.21769/BioProtoc.5438","DOIUrl":"10.21769/BioProtoc.5438","url":null,"abstract":"<p><p>Fusarium crown rot (FCR), mainly caused by <i>Fusarium pseudograminearum</i>, is a devastating soil-borne disease of wheat that results in severe yield and quality reduction. FCR is characterized by stem base necrosis and whitehead development. In recent years, FCR has escalated in both incidence and severity, emerging as a critical threat to global wheat production, particularly within key cultivation zones such as China's Huang-Huai-Hai Plain. The development of resistant cultivars is an effective and environmentally sustainable strategy for FCR disease control. However, the lack of standardized and reproducible inoculation protocols has hindered the accurate assessment and screening of disease-resistant wheat germplasms. To address this limitation, we established a robust FCR inoculation system utilizing <i>F. pseudograminearum</i> propagated on a millet grain substrate, facilitating rapid and reliable evaluation of both host resistance and fungal pathogenicity. Laboratory validation demonstrated high infection efficiency and strong reproducibility of this method. Key features • Standardized and reproducible greenhouse inoculation protocol for Fusarium crown rot resistance assessment, established in wheat. • This system enables accurate quantification of <i>F. pseudograminearum</i> pathogenicity and facilitates high-throughput screening of resistant wheat germplasm.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5438"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457844/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152192","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":"Detection of Autophagy in Human Peripheral Blood Mononuclear Cells Using Guava^® Autophagy and Flow Cytometry.","authors":"Melanie Scherer, Barbara Hochecker, Katja Matt, Alica Meßmer, Jörg Bergemann","doi":"10.21769/BioProtoc.5457","DOIUrl":"10.21769/BioProtoc.5457","url":null,"abstract":"<p><p>Autophagy plays a crucial role in cellular homeostasis and is responsible for removing and degrading damaged cytoplasmic cargo. This lysosome-mediated catabolic process removes defective organelles and misfolded proteins, and impaired autophagy has been directly linked to ageing and numerous diseases. This emphasises the importance of developing intervention methods to counteract this dysregulation. One promising intervention is thermal therapy, specifically hyperthermia, which is described in this protocol. In order to investigate this form of treatment, a rapid and reliable detection method is required to allow comparison of autophagy status under different conditions. While methods such as transmission electron microscopy (TEM) or western blotting can provide valuable structural analysis, they are often time-consuming and expensive, and are not suitable for small, round cells such as peripheral blood mononuclear cells (PBMCs). The method described in this protocol enables absolute quantification of PBMCs using the Guava^® Autophagy Detection kit after heat treatment with water-filtered infrared-A radiation (wIRA), compared with an untreated control. This method is based on antibody labelling, and subsequent flow cytometric analysis enables the number of autophagosomes to be determined by measuring the FITC intensity. This protocol provides rapid, reliable results and can be adapted to investigate not only heat therapy, but also other interventions, such as caloric restriction. Key features • Rapid and reliable ex vivo quantification of autophagy in living cells. • Optimised protocol for the determination of autophagy in primary human blood cells. • Allows the testing of active substances and treatments concerning autophagy. • Flow cytometry-based method for the determination of autophagy.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5457"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457839/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152323","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":"Practical Guide to In Vitro Clonal Propagation of <i>Nicotiana benthamiana</i> Using Axillary Shoot Induction.","authors":"Pradeep Chand Deo","doi":"10.21769/BioProtoc.5443","DOIUrl":"10.21769/BioProtoc.5443","url":null,"abstract":"<p><p>This protocol outlines a reliable method for the micropropagation of <i>Nicotiana benthamiana</i> using axillary shoot branching. Axillary shoot induction involves stimulating the outgrowth of dormant buds located at the leaf axils, allowing for the development of genetically stable shoots without callus formation or the use of exogenous plant growth regulators. Nodal explants are cultured on MS medium supplemented with kinetin and indole-3-butyric acid (IBA) to induce shoot formation. Isolated shoots are then transferred to hormone-free MS medium for rooting. This method is simple, reproducible, and supports rapid plant multiplication for downstream applications such as agroinfiltration or transient protein expression. Key features • Axillary branching-based micropropagation enables efficient regeneration of <i>N. benthamiana</i> from nodal explants, supporting a cyclic system for continuous in vitro plant supply [1]. • Rooting occurs on hormone-free medium, with even shoots as small as 0.5 mm readily forming roots, simplifying and accelerating the propagation process. • Root formation begins within 7 days, including in transgenic lines that are typically difficult to root using conventional methods. • Fully developed, rooted plants are ready within 4 weeks, ideal for transient expression assays, recombinant protein production, and secondary metabolite extraction.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5443"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457845/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152384","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":"Cost-Effective and Reproducible Preparation of mRNA-Loaded Lipid Nanoparticles Using a Conventional Laboratory-Scale Microfluidic Assembly System.","authors":"Yunqi Li, Min Wu, Ruoyang Zhao","doi":"10.21769/BioProtoc.5458","DOIUrl":"10.21769/BioProtoc.5458","url":null,"abstract":"<p><p>This protocol describes a standardized and economically accessible method for synthesizing mRNA-encapsulated lipid nanoparticles using routine laboratory equipment, including precision syringe pumps, Y-shaped glass microfluidic chips, and silicone tubing. Designed to address the cost and accessibility limitations of commercial microfluidic platforms, the system achieves performance metrics comparable to high-end devices while reducing equipment costs by 90%. By systematically optimizing hydrodynamic parameters (total flow rate: 12 mL/min; lipid-to-aqueous phase ratio: 3:1), the protocol enables consistent production of lipid nanoparticles with key quality attributes: high mRNA encapsulation efficiency (≥ 80%), narrow particle size distribution (100-120 nm, polydispersity index ≤ 0.2), and excellent storage performance (≥ 7 days at 4 °C). Key features • A low-cost mRNA@LNPs synthesis method is developed using common lab equipment, cutting costs by 90% while matching commercial systems' performance. • The LNP synthesis platform allows researchers to flexibly adjust hydrodynamic parameters to screen various lipid formulations.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5458"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457847/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152215","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":"Verification of N-Linked Glycosylation of Proteins Isolated from Plant or Mammalian Cell Lines Using PNGase Enzyme.","authors":"Wuqiang Hong, Cong Lei, Yahong Qiu, Yilan Zhou, Yili Hu, Xing Chen, Xilong Li, Jiayang Li","doi":"10.21769/BioProtoc.5444","DOIUrl":"10.21769/BioProtoc.5444","url":null,"abstract":"<p><p>N-glycosylation is a ubiquitous post-translational modification (PTM) that regulates protein folding, stability, and biological function. Accurate identification and validation of N-glycosylation are therefore critical for understanding how glycosylation modulates protein activity. Here, we present a robust workflow for analyzing protein N-glycosylation in both animal and plant systems using peptide-<i>N</i> ⁴-(<i>N</i>-acetyl-β-glucosaminyl) asparagine-amidase A and F (PNGase A and PNGase F). After enzymatic cleavage of the asparagine-linked N-glycans, samples are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting (WB) to detect shifts in apparent molecular weight (MW) indicative of deglycosylation. Key steps include denaturing the protein to expose glycosylation sites, optimizing buffer conditions for PNGase F and A treatment, and comparing glycosylated vs. deglycosylated forms by electrophoretic mobility. A troubleshooting guide addresses common challenges, including incomplete deglycosylation and low transfer efficiency during WB, offering practical solutions to ensure reliable results. This protocol provides researchers with a standardized, cost-effective framework for investigating protein N-glycosylation in diverse systems, from cell lysates to purified proteins, in both animal and plant models. Key features • This method employs standard biochemical techniques, such as enzymatic digestion and SDS-PAGE, making it highly accessible and relatively quick to perform. • Successful deglycosylation results in a detectable downward shift in protein migration on an SDS-PAGE gel. This shift provides a visually confirmable indication of N-glycan removal. • Prior to engaging in mass spectrometry analyses, this approach serves as a rapid, cost-effective preliminary screening or validation tool for assessing N-glycosylation status. • Broad system compatibility: PNGase F and A enzymes are active in mammalian, plant, and microbial systems, allowing reliable N-glycosylation assessment regardless of sample origin.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5444"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152335","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":"A Model of Breast Cancer Micrometastasis in a Three-Dimensional (3D) Liver Spheroid for Testing an Antimetastatic Therapy.","authors":"Kseniya V Nevskaya, Alexandra G Pershina, Lina V Efimova, Ekaterina V Sukhinina, Polina K Kozlova, Alina Yu Ryzhkova, Ekaterina S Hmelevskaya, Marina K Ibragimova, Irina A Tsydenova, Nikolai V Litviakov, Elena V Udut","doi":"10.21769/BioProtoc.5454","DOIUrl":"10.21769/BioProtoc.5454","url":null,"abstract":"<p><p>Even though the survival and proliferation stages of cancer cells that have newly settled at a metastatic site are the rate-limiting stages and the most promising targets for drugs, there is a lack of models of the earliest stage of metastasis formation. A method for modeling breast cancer liver metastasis is described here: a stage of transition of a differentiated tumor cell into a cell actively proliferating in a three-dimensional (3D) liver spheroid. Opposite to existing heterocellular 3D models of metastases, the protocol allows modeling the initial stage of liver colonization by metastatic cells, the so-called \"micrometastases.\" The method includes obtaining a line of fluorescent tumor cells, fluorescence-activated sorting of differentiated cells, preparing a single-cell suspension of liver cells, forming a liver spheroid in an agarose mold, inducing the tumor cell dedifferentiation and proliferation using IL-6, and intravital microscopy of spheroids, with subsequent processing and analysis of fluorescent images in the ImageJ software. The performance of the proposed model was demonstrated using microRNA therapeutics. The ability of a combination of microRNAs to suppress the transition of micrometastasis to macrometastasis in the 3D liver spheroid was confirmed by an immunofluorescent assay of spheroid sections and transcriptome analysis. Key features • The method introduces a 3D model of liver micrometastasis formation using differentiated tumor cells. • The 3D spheroid consists of all the main types of normal liver cells and better reproduces the microenvironment. • The method allows one to evaluate the effectiveness of a drug that blocks the transition of micrometastases to macrometastases. • The model is optimal for studying RNA-based therapeutic agents, as well as prodrugs that require metabolism in the liver for activation.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5454"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457848/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152156","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":"Genetic Engineering of Humanized Telomere Mice.","authors":"Fan Zhang, De Cheng, Kenneth I Porter, Shuwen Wang, Jiyue Zhu","doi":"10.21769/BioProtoc.5445","DOIUrl":"10.21769/BioProtoc.5445","url":null,"abstract":"<p><p>Telomere shortening is a hallmark of human aging, and telomerase regulation plays a critical role in cellular proliferation and replicative senescence. In human cells, telomere length imposes a limit on proliferative potential, a phenomenon known as the Hayflick limit. However, species-specific differences in telomere dynamics and telomerase regulation between humans and mice present challenges to using mice as accurate models for human telomere-related research. To address this limitation, we engineered a humanized telomerase gene (<i>hmTert</i>) in mice by replacing the non-coding sequences within the mouse <i>Tert</i> locus (<i>mTert</i>) with corresponding regulatory sequences from the human <i>TERT</i> gene. Breeding of these genetically modified mice resulted in progressive telomere shortening over successive generations, ultimately reaching human-like lengths (below 10 kb). This protocol outlines the development of this humanized telomere mouse model, referred to as HuT mice, offering a robust platform for studying human telomere biology and aging-related diseases. Key features • This protocol describes methods to increase the success rates of knocking in large genomic fragments (~47 kb) by integrating CRISPR-Cas9 with homologous recombination. • It enables precise engineering of a humanized telomerase gene (<i>hmTert</i>), faithfully recapitulating human TERT regulation and telomere length dynamics in mice.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5445"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457843/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152429","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":"Preparation and Characterization of Lipid Nanoparticles Co-loaded With DNA and Nitro-Oleic Acid.","authors":"Manthan N Patel, Sachchidanand Tiwari, Jacob S Brenner","doi":"10.21769/BioProtoc.5450","DOIUrl":"10.21769/BioProtoc.5450","url":null,"abstract":"<p><p>Lipid nanoparticles (LNPs) are powerful carriers for nucleic acid delivery, but plasmid DNA-loaded LNPs (pDNA-LNPs) have been limited by inflammation and toxicity. We showed that standard pDNA-LNPs activate the cGAS-STING pathway, leading to severe immune responses and mortality in mice. To overcome this, we co-loaded nitro-oleic acid (NOA), an endogenous STING inhibitor, into pDNA-LNPs. NOA-pDNA-LNPs mitigated inflammation, enabled safe in vivo delivery, and supported sustained gene expression for months. Here, we present a detailed protocol for producing and characterizing NOA-pDNA-LNPs to facilitate safer, long-term gene delivery applications. Key features • Provides a step-by-step protocol to produce plasmid DNA-LNPs co-loaded with nitro-oleic acid (NOA), optimized for both in vitro and in vivo applications. • Includes methods for quantitative assessment of DNA and NOA encapsulation efficiencies, particle size, and quality control metrics.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 18","pages":"e5450"},"PeriodicalIF":1.1,"publicationDate":"2025-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12457836/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145152366","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":"Training Mice to Perform Attentional Set-Shifting Under Head Restraint.","authors":"Katarina Kalajzic, John J Stout, Alexander C Mitchell, Timothy Spellman","doi":"10.21769/BioProtoc.5442","DOIUrl":"10.21769/BioProtoc.5442","url":null,"abstract":"<p><p>Cognitive flexibility is a process that involves dynamically adapting behavior to obtain a desired series of outcomes during continuously changing stimulus-response-reward associations. Attentional set-shifting is a multi-modal decision-making paradigm that tests cognitive flexibility, which can be useful for investigating neural circuitry that is disrupted in multiple neuropsychiatric disorders, including schizophrenia, Alzheimer's disease, depression, and addiction. The canonical human attentional set-shifting paradigm for measuring cognitive flexibility is the Wisconsin Card Sorting Test, in which subjects sort cards based on rules that change periodically and adapt their behavior by utilizing feedback in the form of correct and incorrect outcomes. To transfer these tests to rodent models, previous techniques involved attentional set-shifting paradigms in free-roaming test chambers, in which animals associated cues with reward locations. The protocol presented here involves an analogous attentional set-shifting paradigm, in which water-restricted mice are head-restrained on a platform and must keep track of periodically switching stimulus-response-outcome associations. The mice must learn to associate odor or whisker vibration cues with a binary directional lick response that triggers water delivery. The mice are trained to respond by licking one of two spouts, in which the correct decision is dependent on the current stimulus rule. This protocol allows for behaviorally measuring cognitive flexibility alongside neural activity by pairing the head-restrained paradigm with 2-photon calcium imaging, optogenetics, and extracellular and intracellular physiology. Key features • Head-restraint design that allows for optogenetic manipulation, in vivo imaging, extracellular and intracellular electrophysiology, and other manipulations. • Attentional set-shifting paradigm that measures cognitive flexibility in rodents.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 17","pages":"e5442"},"PeriodicalIF":1.1,"publicationDate":"2025-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12423282/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145066839","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}