Bio-protocol最新文献

{"title":"SIMBA Method-Simultaneous Detection of Antimicrobial and Anti-biofilm Activity of New Compounds Using <i>Salmonella</i> Infantis.","authors":"Meta Sterniša,&nbsp;Jerica Sabotič,&nbsp;Nika Janež,&nbsp;Tomaž Curk,&nbsp;Anja Klančnik","doi":"10.21769/BioProtoc.4783","DOIUrl":"https://doi.org/10.21769/BioProtoc.4783","url":null,"abstract":"The development of antimicrobial resistance and the formation of Salmonella biofilms are serious public health problems. For this reason, new natural compounds with antimicrobial and anti-biofilm activity are being sought, and wild fungi represent an untapped potential. Various extraction agents, including organic solvents and aqueous buffers, can be used to obtain bioactive compounds from natural sources. To evaluate their bioactivity, extensive screening studies are required to determine antimicrobial and anti-biofilm activity using methods such as broth microdilution or crystal violet assay, respectively, but none of these methods allow simultaneous evaluation of both activities against bacteria. Cold water extraction from wild fungi offers the advantage of extracting water-soluble compounds. The SIMultaneous detection of antiMicrobial and anti-Biofilm Activity (SIMBA) method combines the testing of both types of activity against bacteria with the evaluation of the 20 h growth curve of the Salmonella Infantis ŽM9 strain determined with absorbance measurements at 600 nm in a 96-well plate. SIMBA method thus shortens the time to determine the bioactivity of extracts, reduces material consumption, and eliminates the need for additional reagents. SIMBA enables rapid selection of bioactive extracts for their fractionation and shortens the time to determine new natural products with antimicrobial and anti-biofilm activity. Graphical overview","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 15","pages":"e4783"},"PeriodicalIF":0.0,"publicationDate":"2023-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/c4/b7/BioProtoc-13-15-4783.PMC10415211.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9998479","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 qPCR Method to Distinguish between Expression of Transgenic and Endogenous Copies of Genes.","authors":"William Bezodis,&nbsp;Helen Prescott,&nbsp;Daniela Vlad,&nbsp;Hugh Dickinson","doi":"10.21769/BioProtoc.4784","DOIUrl":"https://doi.org/10.21769/BioProtoc.4784","url":null,"abstract":"<p><p>Study of gene function in eukaryotes frequently requires data on the impact of the gene when it is expressed as a transgene, such as in ectopic or overexpression studies. Currently, the use of transgenic constructs designed to achieve these aims is often hampered by the difficulty in distinguishing between the expression levels of the endogenous gene and its transgene equivalent, which may involve either laborious microdissection to isolate specific cell types or harvesting tissue at narrow timepoints. To address this challenge, we have exploited a feature of the Golden Gate cloning method to develop a simple, restriction digest-based protocol to differentiate between expression levels of transgenic and endogenous gene copies. This method is straightforward to implement when the endogenous gene contains a <i>Bpi1</i> restriction site but, importantly, can be adapted for most genes and most other cloning strategies. Key features This protocol was developed to determine the expression level of an ectopically expressed transcription factor with broad native expression in all surrounding tissues. The method described is most directly compatible with Golden Gate cloning but is, in principle, compatible with any cloning method. The protocol has been developed and validated in the model plant <i>Arabidopsis thaliana</i> but is applicable to most eukaryotes. Graphical overview.</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 15","pages":"e4784"},"PeriodicalIF":0.0,"publicationDate":"2023-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/dd/c9/BioProtoc-13-15-4784.PMC10415200.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9998476","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":"Using Fiber Photometry in Mice to Estimate Fluorescent Biosensor Levels During Sleep.","authors":"Mie Andersen,&nbsp;Anastasia Tsopanidou,&nbsp;Tessa Radovanovic,&nbsp;Viviane Noelani Compere,&nbsp;Natalie Hauglund,&nbsp;Maiken Nedergaard,&nbsp;Celia Kjaerby","doi":"10.21769/BioProtoc.4734","DOIUrl":"https://doi.org/10.21769/BioProtoc.4734","url":null,"abstract":"<p><p>Sleep is not homogenous but contains a highly diverse microstructural composition influenced by neuromodulators. Prior methods used to measure neuromodulator levels in vivo have been limited by low time resolution or technical difficulties in achieving recordings in a freely moving setting, which is essential for natural sleep. In this protocol, we demonstrate the combination of electroencephalographic (EEG)/electromyographic (EMG) recordings with fiber photometric measurements of fluorescent biosensors for neuromodulators in freely moving mice. This allows for real-time assessment of extracellular neuromodulator levels during distinct phases of sleep with a high temporal resolution.</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 15","pages":"e4734"},"PeriodicalIF":0.0,"publicationDate":"2023-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/fe/33/BioProtoc-13-15-4734.PMC10415158.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10000522","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":"Fabrication of Microfluidic Devices for Continuously Monitoring Yeast Aging.","authors":"Richard O'Laughlin, Emerald Forrest, Jeff Hasty, Nan Hao","doi":"10.21769/BioProtoc.4782","DOIUrl":"10.21769/BioProtoc.4782","url":null,"abstract":"<p><p>For several decades, aging in <i>Saccharomyces cerevisiae</i> has been studied in hopes of understanding its causes and identifying conserved pathways that also drive aging in multicellular eukaryotes. While the short lifespan and unicellular nature of budding yeast has allowed its aging process to be observed by dissecting mother cells away from daughter cells under a microscope, this technique does not allow continuous, high-resolution, and high-throughput studies to be performed. Here, we present a protocol for constructing microfluidic devices for studying yeast aging that are free from these limitations. Our approach uses multilayer photolithography and soft lithography with polydimethylsiloxane (PDMS) to construct microfluidic devices with distinct single-cell trapping regions as well as channels for supplying media and removing recently born daughter cells. By doing so, aging yeast cells can be imaged at scale for the entirety of their lifespans, and the dynamics of molecular processes within single cells can be simultaneously tracked using fluorescence microscopy. Key features This protocol requires access to a photolithography lab in a cleanroom facility. Photolithography process for patterning photoresist on silicon wafers with multiple different feature heights. Soft lithography process for making PDMS microfluidic devices from silicon wafer templates.</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 15","pages":"e4782"},"PeriodicalIF":0.0,"publicationDate":"2023-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10415209/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10000521","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":"Bi-directional Dual-flow-RootChip for Physiological Analysis of Plant Primary Roots Under Asymmetric Perfusion of Stress Treatments.","authors":"Claudia Allan,&nbsp;Blake Elliot,&nbsp;Volker Nock,&nbsp;Claudia-Nicole Meisrimler","doi":"10.21769/BioProtoc.4764","DOIUrl":"https://doi.org/10.21769/BioProtoc.4764","url":null,"abstract":"<p><p>Due to technical limitations, research to date has mainly focused on the role of abiotic and biotic stress-signalling molecules in the aerial organs of plants, including the whole shoot, stem, and leaves. Novel experimental platforms including the dual-flow-RootChip (dfRC), PlantChip, and RootArray have since expanded this to plant-root cell analysis. Based on microfluidic platforms for flow stream shaping and force sensing on tip-growing organisms, the dfRC has further been expanded into a bi-directional dual-flow-RootChip (bi-dfRC), incorporating a second adjacent pair of inlets/outlet, enabling bi-directional asymmetric perfusion of treatments towards plant roots (shoot-to-root or root-to-shoot). This protocol outlines, in detail, the design and use of the bi-dfRC platform. Plant culture on chip is combined with guided root growth and controlled exposure of the primary root to solute changes. The impact of surface treatment on root growth and defence signals can be tracked in response to abiotic and biotic stress or the combinatory effect of both. In particular, this protocol highlights the ability of the platform to culture a variety of plants, such as <i>Arabidopsis thaliana</i>, <i>Nicotiana benthamiana</i>, and <i>Solanum lycopersicum</i>, on chip. It demonstrates that by simply altering the dimensions of the bi-dfRC, a broad application basis to study desired plant species with varying primary root sizes under microfluidics is achieved. Key features Expansion of the method developed by Stanley et al. (2018a) to study the directionality of defence signals responding to localised treatments. Description of a microfluidic platform allowing culture of plants with primary roots up to 40 mm length, 550 μm width, and 500 μm height. Treatment with polyvinylpyrrolidone (PVP) to permanently retain the hydrophilicity of partially hydrophobic bi-dfRC microchannels, enabling use with surface-sensitive plant lines. Description of novel tubing array setup equipped with rotatable valves for switching treatment reagent and orientation, while live-imaging on the bi-dfRC. Graphical overview <b>Graphical overview of bi-dfRC fabrication, plantlet culture, and setup for root physiological analysis.</b>(a) Schematic diagram depicting photolithography and replica molding, to produce a PDMS device. (b) Schematic diagram depicting seed culture off chip, followed by sub-culture of 4-day-old plantlets on chip. (c) Schematic diagram depicting microscopy and imaging setup, equipped with a media delivery system for asymmetric treatment introduction into the bi-dfRC microchannel root physiological analysis under varying conditions.</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 15","pages":"e4764"},"PeriodicalIF":0.0,"publicationDate":"2023-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f1/b5/BioProtoc-13-15-4764.PMC10415191.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9998477","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":"Engineering <i>Agrobacterium tumefaciens</i> with a Type III Secretion System to Express Type III Effectors.","authors":"Vidhyavathi Raman,&nbsp;Kirankumar S Mysore","doi":"10.21769/BioProtoc.4726","DOIUrl":"https://doi.org/10.21769/BioProtoc.4726","url":null,"abstract":"<p><p>Plants elicit defense responses when exposed to pathogens, which partly contribute to the resistance of plants to Agrobacterium tumefaciens-mediated transformation. Some pathogenic bacteria have sophisticated mechanisms to counteract these defense responses by injecting Type III effectors (T3Es) through the Type III secretion system (T3SS). By engineering <i>A. tumefaciens</i> to express T3SS to deliver T3Es, we suppressed plant defense and enhanced plant genetic transformation. Here, we describe the optimized protocols for mobilization of T3SS-expressing plasmid to engineer <i>A. tumefaciens</i> to deliver proteins through T3SS and fractionation of cultures to study proteins from pellet and supernatants to determine protein secretion from engineered <i>A. tumefaciens</i>.</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 15","pages":"e4726"},"PeriodicalIF":0.0,"publicationDate":"2023-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/c7/68/BioProtoc-13-15-4726.PMC10415197.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10052283","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":"Construction of Activity-based Anorexia Mouse Models.","authors":"Maria Consolata Miletta, Tamas L Horvath","doi":"10.21769/BioProtoc.4730","DOIUrl":"10.21769/BioProtoc.4730","url":null,"abstract":"<p><p>Anorexia nervosa (AN) is a psychiatric disorder mainly characterized by extreme hypophagia, severe body weight loss, hyperactivity, and hypothermia. Currently, AN has the highest mortality rate among psychiatric illnesses. Despite decades of research, there is no effective cure for AN nor is there a clear understanding of its etiology. Since a complex interaction between genetic, environmental, social, and cultural factors underlines this disorder, the development of a suitable animal model has been difficult so far. Here, we present our protocol that couples a loss-of-function mouse model to the activity-based anorexia model (ABA), which involves self-imposed starvation in response to exposure to food restriction and exercise. We provide insights into a neural circuit that drives survival in AN and, in contrast to previous protocols, propose a model that mimics the conditions that mainly promote AN in humans, such as increased incidence during adolescence, onset preceded by negative energy balance, and increased compulsive exercise. This protocol will be useful for future studies that aim to identify neuronal populations or brain circuits that promote the onset or long-term maintenance of this devastating eating disorder.</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 15","pages":"e4730"},"PeriodicalIF":0.0,"publicationDate":"2023-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/29/45/BioProtoc-13-15-4730.PMC10415190.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9995305","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":"Intravital Imaging of Intestinal Intraepithelial Lymphocytes.","authors":"Sara McArdle, Goo-Young Seo, Mitchell Kronenberg, Zbigniew Mikulski","doi":"10.21769/BioProtoc.4720","DOIUrl":"10.21769/BioProtoc.4720","url":null,"abstract":"<p><p>Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol of intravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α⁺ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 14","pages":"e4720"},"PeriodicalIF":0.0,"publicationDate":"2023-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f4/33/BioProtoc-13-14-4720.PMC10366999.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9937129","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":"Visualizing Loss of Plasma Membrane Lipid Asymmetry Using Annexin V Staining.","authors":"Julia F Baum,&nbsp;Huriye D Uzun,&nbsp;Thomas Günther Pomorski","doi":"10.21769/BioProtoc.4754","DOIUrl":"https://doi.org/10.21769/BioProtoc.4754","url":null,"abstract":"<p><p>Loss of plasma membrane lipid asymmetry contributes to many cellular functions and responses, including apoptosis, blood coagulation, and cell fusion. In this protocol, we describe the use of fluorescently labeled annexin V to detect loss of lipid asymmetry in the plasma membrane of adherent living cells by fluorescence microscopy. The approach provides a simple, sensitive, and reproducible method to detect changes in lipid asymmetry but is limited by low sample throughput. The protocol can also be adapted to other fluorescently labeled lipid-binding proteins or peptide probes. To validate the lipid binding properties of such probes, we additionally describe here the preparation and use of giant unilamellar vesicles as simple model membrane systems that have a size comparable to cells. Key features Monitoring loss of lipid asymmetry in the plasma membrane via confocal microscopy. Protocol can be applied to any type of cell that is adherent in culture, including primary cells. Assay can be adapted to other fluorescently labeled lipid-binding proteins or peptide probes. Giant unilamellar vesicles serve as a tool to validate the lipid binding properties of such probes. Graphical overview <b>Imaging the binding of fluorescent annexin V to adherent mammalian cells and giant vesicles by confocal microscopy.</b> Annexin V labeling is a useful method for detecting a loss of plasma membrane lipid asymmetry in cells (top image, red); DAPI can be used to identify nuclei (top image, blue). Giant vesicles are used as a tool to validate the lipid binding properties of annexin V to anionic lipids (lower image, red).</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 14","pages":"e4754"},"PeriodicalIF":0.0,"publicationDate":"2023-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/b2/bd/BioProtoc-13-14-4754.PMC10366992.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10258894","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":"Optogenetic Induction of Pyroptosis, Necroptosis, and Apoptosis in Mammalian Cell Lines.","authors":"Kateryna Shkarina,&nbsp;Petr Broz","doi":"10.21769/BioProtoc.4762","DOIUrl":"https://doi.org/10.21769/BioProtoc.4762","url":null,"abstract":"<p><p>Regulated cell death plays a key role in immunity, development, and homeostasis, but is also associated with a number of pathologies such as autoinflammatory and neurodegenerative diseases and cancer. However, despite the extensive mechanistic research of different cell death modalities, the direct comparison of different forms of cell death and their consequences on the cellular and tissue level remain poorly characterized. Comparative studies are hindered by the mechanistic and kinetic differences between cell death modalities, as well as the inability to selectively induce different cell death programs in an individual cell within cell populations or tissues. In this method, we present a protocol for rapid and specific optogenetic activation of three major types of programmed cell death: apoptosis, necroptosis, and pyroptosis, using light-induced forced oligomerization of their major effector proteins (caspases or kinases).</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 14","pages":"e4762"},"PeriodicalIF":0.0,"publicationDate":"2023-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10366993/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10241468","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}