Current protocols最新文献_第3页

DMS-MapSeq Analysis of Antisense Oligonucleotide Binding to lncRNA PANDA 反义寡核苷酸与 lncRNA PANDA 结合的 DMS-MapSeq 分析。

Current protocols Pub Date : 2024-11-26 DOI: 10.1002/cpz1.70038

Gabriel A. Romero Agosto, Ethan Cox, Silvi Rouskin

引用次数: 0

Human Pancreas-Derived Organoids with Controlled Polarity: Detailed Protocols and Experimental Timeline 具有可控极性的人胰腺衍生有机体：详细规程和实验时间表。

Current protocols Pub Date : 2024-11-26 DOI: 10.1002/cpz1.70045

Aletta Kiss, Attila Farkas, Ferhan Ayaydin, György Lázár, Árpád Varga, József Maléth

{"title":"Human Pancreas-Derived Organoids with Controlled Polarity: Detailed Protocols and Experimental Timeline","authors":"Aletta Kiss, Attila Farkas, Ferhan Ayaydin, György Lázár, Árpád Varga, József Maléth","doi":"10.1002/cpz1.70045","DOIUrl":"10.1002/cpz1.70045","url":null,"abstract":"Since their discovery, 3D cell cultures have emerged as powerful tools across various basic, translational research, and industrial discovery projects. One such application is in the physiological and pathophysiological modeling of pancreatic exocrine functions, which addresses critical clinical challenges, including acute and chronic pancreatitis. While several methods now exist for generating epithelial organoids (derived from induced pluripotent, embryonic, or adult stem cells), the advent of patient-derived organoids (PDOs) with controlled polarity has introduced a new frontier in pancreatic research. This advancement has significantly expanded the methodological arsenal available for studying human pancreatic epithelial secretion.In this article, we present basic protocols and a troubleshooting guide for an advanced culture method that results in an apical-to-basal polarity switch. Alongside the protocols, we emphasize a comprehensive cost breakdown, an aspect often challenging to estimate when implementing new techniques. By sharing the technical nuances and financial implications of these protocols, we aim to encourage researchers to transition from rodent models to primary human epithelial cells wherever feasible. This aligns with the U.S. Environmental Protection Agency's efforts to accelerate the translation of significant scientific findings to address major clinical needs. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Establishment and maintenance of pancreatic PDOsBasic Protocol 2: Cryopreservation and thawing of pancreatic PDOsBasic Protocol 3: Inducing polarity switching in pancreatic PDOs","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142717865","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}

引用次数: 0

Multi-site Ultrasound-guided Fine Needle Aspiration to Study Cells and Soluble Factors From Human Lymph Nodes 多部位超声引导细针抽吸法研究人体淋巴结的细胞和可溶性因子

Current protocols Pub Date : 2024-11-23 DOI: 10.1002/cpz1.70063

Adam Al-Diwani, Deepsha Agrawal, Fintan Sheerin, Callum Board, Sarosh R. Irani, Katrina M. Pollock, Nicholas M. Provine

{"title":"Multi-site Ultrasound-guided Fine Needle Aspiration to Study Cells and Soluble Factors From Human Lymph Nodes","authors":"Adam Al-Diwani, Deepsha Agrawal, Fintan Sheerin, Callum Board, Sarosh R. Irani, Katrina M. Pollock, Nicholas M. Provine","doi":"10.1002/cpz1.70063","DOIUrl":"10.1002/cpz1.70063","url":null,"abstract":"Lymph nodes (LNs) are specialized secondary lymphoid tissues essential to the priming and maintenance of adaptive immune responses, including the B cell germinal center response; thus, they are central to immunity. However, the anatomically restricted and time-resolved nature of immune priming means that sampling disease-relevant human LNs requires specialized techniques. This article describes the application of ultrasound-guided fine-needle aspiration (FNA) to sample LNs, using cervical LNs of the head and neck as an exemplar. This minimally invasive technique allows collection of both immune cells and cell-free material that are relevant to both neuroimmune diseases and basic lymphatic functions. Downstream use of cellular material can include multiplexed flow cytometry, single-cell transcriptome sequencing (RNA-seq), and B cell cultures. The cell-free supernatant can be used for proteomics or other similar ‘omics approaches. This unit describes collection of samples by FNA as well as processing and storage of samples for downstream assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Sampling of human cervical lymph nodes by ultrasound-guided fine-needle aspirationAlternate Protocol: Sampling of human lymph nodes by ultrasound-guided fine-needle aspiration with negative pressureBasic Protocol 2: Processing and storage of human lymph node samples","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70063","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142695792","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}

引用次数: 0

Analysis of Free Oligosaccharides in Urine by High-Performance Liquid Chromatography–Tandem Mass Spectrometry 利用高效液相色谱-串联质谱法分析尿液中的游离低聚糖

Current protocols Pub Date : 2024-11-22 DOI: 10.1002/cpz1.70055

J. Daniel Sharer, Rongrong Huang, Timothy Wood, Paula Huffman, Yang Yan, Chelsea Zimmerman, Laura Pollard

引用次数: 0

Synthesis and Application of a Caged Bioluminescent Probe for the Immunoproteasome 免疫蛋白酶体笼式生物发光探针的合成与应用

Current protocols Pub Date : 2024-11-22 DOI: 10.1002/cpz1.70057

Cody A. Loy, Darci J. Trader

{"title":"Synthesis and Application of a Caged Bioluminescent Probe for the Immunoproteasome","authors":"Cody A. Loy, Darci J. Trader","doi":"10.1002/cpz1.70057","DOIUrl":"10.1002/cpz1.70057","url":null,"abstract":"Monitoring the catalytic activity of the proteasome and its various isoforms has become increasingly important with the continued development of core particle inhibitors and targeted protein degraders as potential therapies for diseases with high protein accumulation. The immunoproteasome (iCP) is expressed in a variety of diseases due to inflammatory signals, such as interferon-gamma, that alert the cell to begin generating iCP preferentially over the standard proteasome. There is a need to understand iCP activity and expression both in cells and in vivo because it is becoming a widely targeted isoform in a variety of diseases. Activity-based probes for the iCP have been developed, but their application has been limited due to their difficult synthesis and choice of fluorescent reporter. There has yet to be a selective iCP probe developed that incorporates a luminescent reporter that could be applied to a variety of different applications. The protocols presented here describe the synthesis of a cleavable activity-based bioluminescent probe that is selective for the iCP, and the application of the synthesized probe in immunoproteasome activity assays using a luminescent plate reader. Having this bioluminescent reporter, a better understanding of how the iCP is implicated in disease progression, as well as identification of small molecule interactors, can be achieved. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Synthesis of a bioluminescent immunoproteasome probeBasic Protocol 2: Expression of the immunoproteasome in cellsBasic Protocol 3: Immunoproteasome probe application in live cells using a luminescent plate reader","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142690025","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}

引用次数: 0

Workflow to Select Functional Promoter DNA Baits and Screen Arrayed Gene Libraries in Yeast 在酵母中选择功能性启动子 DNA 诱饵和筛选阵列基因库的工作流程。

Current protocols Pub Date : 2024-11-21 DOI: 10.1002/cpz1.70059

Iris Fañanás-Pueyo, Ana-Mariya Anhel, Ángel Goñi-Moreno, Luis Oñate-Sánchez, Gerardo Carrera-Castaño

{"title":"Workflow to Select Functional Promoter DNA Baits and Screen Arrayed Gene Libraries in Yeast","authors":"Iris Fañanás-Pueyo, Ana-Mariya Anhel, Ángel Goñi-Moreno, Luis Oñate-Sánchez, Gerardo Carrera-Castaño","doi":"10.1002/cpz1.70059","DOIUrl":"10.1002/cpz1.70059","url":null,"abstract":"The yeast one-hybrid system (Y1H) is used extensively to identify DNA–protein interactions. The generation of large collections of open reading frames (ORFs) to be used as prey in screenings is not a bottleneck nowadays and can be carried out in-house or offered as a service by companies. However, the straightforward use of full gene promoters as baits to identify interacting proteins undermines the accuracy and sensitivity of the assay, especially in the case of multicellular eukaryotes. Therefore, it is paramount to implement procedures for efficient identification of suitable promoter fragments compatible with the Y1H assay. Here, we describe a workflow to identify biologically relevant conserved promoter fragments of Arabidopsis thaliana through simple and robust phylogenetic analyses. Additionally, we describe a manual method and its automated robotized version for rapid and efficient high-throughput Y1H screenings of arrayed ORF libraries with the identified DNA fragments. Moreover, this method can be scaled up or down and used for yeast two-hybrid screenings to search for possible interactors of proteins identified by the Y1H approach or any other protein of interest, altogether underscoring its suitability to build gene regulatory networks. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Selection of DNA baits for Y1H screeningsBasic Protocol 2: Y1H screenings with arrayed gene librariesAlternate Protocol: Automated screening with a liquid-handling robot","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70059","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142683995","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}

引用次数: 0

Vesicular Stomatitis Virus as a Platform for Protease Activity Measurements 作为蛋白酶活性测量平台的水泡性口炎病毒

Current protocols Pub Date : 2024-11-21 DOI: 10.1002/cpz1.70062

Stefanie Rauch, Francesco Costacurta, Dorothee von Laer, Emmanuel Heilmann

{"title":"Vesicular Stomatitis Virus as a Platform for Protease Activity Measurements","authors":"Stefanie Rauch, Francesco Costacurta, Dorothee von Laer, Emmanuel Heilmann","doi":"10.1002/cpz1.70062","DOIUrl":"10.1002/cpz1.70062","url":null,"abstract":"Protease inhibitors are among the most powerful antiviral drugs. They have been used successfully against viruses, such as the human immunodeficiency virus (HIV), hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protease inhibitor screening tools are therefore important to identify inhibitors that have the potential to become antiviral drugs. In this article, we describe newly developed cell- and virus replicon-based platforms to screen inhibitors. We developed the methods presented here by genetically modifying vesicular stomatitis virus, a model virus from the family Rhabdoviridae. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Mpro-On and -Off assayAlternate Protocol 1: Virus production with transient P- and L TransIT transfectionAlternate Protocol 2: Virus production with transient P- and L Ca2PO4 transfectionAlternate Protocol 3: Luciferase-based variation of the On assayAlternate Protocol 4: Screening assay with fluorescence-activated cell sorting readoutSupport Protocol: Performing kinetic measurements with Off assay","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70062","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142683993","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}

引用次数: 0

Engineering and Evaluating Vascularized Organotypic Spheroids On-Chip 芯片上血管有组织球体的工程设计与评估

Current protocols Pub Date : 2024-11-21 DOI: 10.1002/cpz1.70058

James J. Tronolone, Nadin Mohamed, Christopher P. Chaftari, Yuxiang Sun, Tanmay Mathur, Abhishek Jain

{"title":"Engineering and Evaluating Vascularized Organotypic Spheroids On-Chip","authors":"James J. Tronolone, Nadin Mohamed, Christopher P. Chaftari, Yuxiang Sun, Tanmay Mathur, Abhishek Jain","doi":"10.1002/cpz1.70058","DOIUrl":"10.1002/cpz1.70058","url":null,"abstract":"Organotypic spheroids are evolving as a mainstream in vitro modeling platform, but it is crucial to integrate vascular tissue and perfusion for maintaining their longevity, stability, and physiological relevance. Current vascularization methods remain underdeveloped, and several protocols are poorly reproducible and are limited to use by a few select groups who have designed these methods. To achieve standardization, we offer a step-by-step guide to vascularize organotypic spheroids in case studies of pancreatic islets and cancer spheroids. Our systematic approach spans microfluidic chip design, spheroid fabrication, and vascularization techniques (vasculogenesis and angiogenesis) while describing critical tissue engineering methods. We also include additional insights and operating guidelines within our protocols that characterize and quantitate these models with molecular assays as well as our integrated computational algorithms of mass transport through formed capillary vessels. These protocols contribute to establishing reproducibility, standardization, and enhanced adoption by other contemporary organ-chip researchers, who want to engineer vascularized organoid-based microphysiological platforms. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Design and fabrication of microfluidic chips for vascularized spheroidsBasic Protocol 2: Organotypic spheroid fabricationBasic Protocol 3: Vascularized spheroids on-chipBasic Protocol 4: Functionality assaysSupport Protocol 1: Cell CultureSupport Protocol 2: Immunocytochemistry","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142683991","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}

引用次数: 0

Injection of Ortho-Functionalized Tetrafluorinated Azobenzene-Containing siRNAs into Japanese Medaka Embryos for Photocontrolled Gene Silencing 向日本青鳉胚胎注射含 siRNA 的正功能化四氟偶氮苯，实现光控基因沉默。

Current protocols Pub Date : 2024-11-15 DOI: 10.1002/cpz1.70051

Makenzie Mateus, Matthew L. Hammill, Denina B. D. Simmons, Jean-Paul Desaulniers

{"title":"Injection of Ortho-Functionalized Tetrafluorinated Azobenzene-Containing siRNAs into Japanese Medaka Embryos for Photocontrolled Gene Silencing","authors":"Makenzie Mateus, Matthew L. Hammill, Denina B. D. Simmons, Jean-Paul Desaulniers","doi":"10.1002/cpz1.70051","DOIUrl":"10.1002/cpz1.70051","url":null,"abstract":"This article describes the detailed methodology of how to inject photoswitchable ortho-functionalized tetrafluorinated short interfering RNAs (F-siRNAs) into a single cell of stage-two Japanese medaka (Oryzias latipes) embryos and how to control gene silencing with different wavelengths of light. Many of the prior papers describing Japanese medaka embryo injections omit key information. As such, this article aims to give an in-depth explanation as to how the NanoJect III microinjector can be used for this purpose. To obtain the embryos for microinjection, adult medaka are housed under a 14-hr light, 10-hr dark cycle to mimic their natural breeding period. This induces mating at approximately the same time each day, when the lights turn on, so recently fertilized eggs can be obtained. Synthetic F-siRNAs are injected into transgenic stage-two single-cell Japanese medaka embryos expressing enhanced green fluorescent protein (eGFP). Our data demonstrate that our F-siRNAs can silence gene activity in Japanese medaka embryos expressing eGFP. Moreover, gene expression can be activated by exposing F-siRNA-injected embryos to blue light and deactivated a few days after exposure to green light. To the best of our knowledge, this marks the first reversible control of a gene-silencing oligonucleotide within an in vivo system. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Medaka maintenance and embryo collectionBasic Protocol 2: Injection of stage-two one-cell medaka embryosBasic Protocol 3: Evaluation of the F-siRNA gene-silencing ability through light activation and inactivation using blue and green light by measuring enhanced green fluorescent protein fluorescence","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.70051","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142639242","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}

引用次数: 0

Generating iAstrocytes From Human Induced Pluripotent Stem Cells by Combining Low-Density Passaging of Neural Progenitor Cells and Transcription Factor NFIA Transdifferentiation 将神经祖细胞的低密度传代与转录因子 NFIA 的转分化相结合，从人类诱导多能干细胞中生成 iAstrocytes。

Current protocols Pub Date : 2024-11-15 DOI: 10.1002/cpz1.70049

Patrick Bosco, Ugur Akcan, Damian Williams, Heather M. Buchanan, Dritan Agalliu, Andrew A. Sproul

{"title":"Generating iAstrocytes From Human Induced Pluripotent Stem Cells by Combining Low-Density Passaging of Neural Progenitor Cells and Transcription Factor NFIA Transdifferentiation","authors":"Patrick Bosco, Ugur Akcan, Damian Williams, Heather M. Buchanan, Dritan Agalliu, Andrew A. Sproul","doi":"10.1002/cpz1.70049","DOIUrl":"10.1002/cpz1.70049","url":null,"abstract":"Astrocytes are key regulators of central nervous system (CNS) homeostasis, and their dysfunction is implicated in neurological and neurodegenerative disorders. Here, we describe a two-step protocol to generate astrocytes from human induced pluripotent stem cells (hiPSCs) using a bankable neural progenitor cell (NPC) intermediate, followed by low-density passaging and overexpression of the gliogenic transcription factor NFIA. A bankable NPC intermediate allows for facile differentiation into both purified neuronal and astrocyte cell types in parallel from the same genetic background, depending on the experimental needs. This article presents a protocol to generate NPCs from hiPSCs, which are then differentiated into hiPSC-derived astrocytes, termed iAstrocytes. The resulting iAstrocytes express key markers of astrocyte identity at transcript and protein levels by bulk RNA-Seq and immunocytochemistry, respectively. Additionally, they respond to the inflammatory stimuli poly(I:C) and generate waves of calcium activity in response to either physical activity or the addition of ATP. Our approach offers a simple and robust method to generate and characterize human astrocytes, which can be used to model human disease affecting this cell type. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Differentiation of hiPSCs to NPCsBasic Protocol 2: Differentiation of NPCs into iAstrocytesSupport Protocol 1: Molecular validation of iAstrocytesSupport Protocol 2: Calcium imaging-based validation of iAstrocyte functionSupport Protocol 3: Differentiation of NPCs into neurons","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142638765","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}

引用次数: 0