Springer Protocols platform has migrated to Experiments

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class="control__indicator" tabindex="0"></div> </li> <li class="facet-value at-3 control"> <input class="facet-checkbox" value="Escherichia coli" name="organismFacet" id="organism-facet_Escherichia_coli" type="checkbox" autocomplete="off" tabindex="0"> <label class="facet-value-title" for="organism-facet_Escherichia_coli">Escherichia coli</label> <span class="facet-value-count">6906</span> <div class="control__indicator" tabindex="0"></div> </li> <li class="facet-value at-4 control"> <input class="facet-checkbox" value="Bos taurus" name="organismFacet" id="organism-facet_Bos_taurus" type="checkbox" autocomplete="off" tabindex="0"> <label class="facet-value-title" for="organism-facet_Bos_taurus">Bos taurus</label> <span class="facet-value-count">3796</span> <div class="control__indicator" tabindex="0"></div> </li> <li class="facet-value at-5 control"> <input class="facet-checkbox" value="Rattus norvegicus" name="organismFacet" id="organism-facet_Rattus_norvegicus" type="checkbox" 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class="control__indicator" tabindex="0"></div> </li> <li class="facet-value at-8 control"> <input class="facet-checkbox" value="Arabidopsis" name="organismFacet" id="organism-facet_Arabidopsis" type="checkbox" autocomplete="off" tabindex="0"> <label class="facet-value-title" for="organism-facet_Arabidopsis">Arabidopsis</label> <span class="facet-value-count">1127</span> <div class="control__indicator" tabindex="0"></div> </li> <li class="facet-value at-9 control"> <input class="facet-checkbox" value="Arabidopsis thaliana" name="organismFacet" id="organism-facet_Arabidopsis_thaliana" type="checkbox" autocomplete="off" tabindex="0"> <label class="facet-value-title" for="organism-facet_Arabidopsis_thaliana">Arabidopsis thaliana</label> <span class="facet-value-count">979</span> <div class="control__indicator" tabindex="0"></div> </li> </ol> </div> </div> <div class="SN_search-Results__facet" id="cellline" style="display:block"> <div class="SN_search-Results__facets-header"> <h2>Cell 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class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4140-8"><span class="italics">Glaucoma</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4140-8_16, Springer Protocols, 1' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4140-8_16'>Retinal Ganglion Cell Subtypes and Their Vulnerability in Glaucoma</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">James E. Morgan <a class="author-email" href="mailto:morganje3@cardiff.ac.uk"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> <span class="comma_space">, </span> <span class="author-name">Ryan J. Bevan </span> <span class="comma_space">, </span> <span class="author-name">Gloria Cimaglia </span> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>The detection of selective retinal ganglion cell damage in glaucoma has been a long sought-after goal, not just for the development of clinical tests for the early detection of glaucoma but for the elucidation of potential mechanisms underlying</p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4140-8_16'>more</a> </span> </div> <div class="snippet-details hidden"><p>The detection of selective retinal ganglion cell damage in glaucoma has been a long sought-after goal, not just for the development of clinical tests for the early detection of glaucoma but for the elucidation of potential mechanisms underlying retinal ganglion cell loss. Early reports of the selective vulnerability of larger retinal ganglion cells (RGCs) in human studies did not translate simply to the loss of a particular class of RGC but more likely reflected shrinkage and degeneration across all RGC classes. Subsequent studies of nonhuman primate (NHP) models of glaucoma indicated some selectivity with great damage to the magnocellular vs parvocellular pathways. More recently, rodent models of experimental glaucoma have highlighted a selective vulnerability of OFF-centered RGCs—particularly those with transient responses. Selectivity for OFF pathway damage is also seen as a trend in a rat model of glaucoma. These data support the concept that some RGCs are more vulnerable to the effects of glaucoma damage. This chapter covers some of the methods to elucidate RGC damage and the relevance of model selection to mimic human glaucoma rather than just RGC death.</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p><span>Techniques: </span>Diagnostic Techniques, Dissection</p> </div> </div> <div class="snippet-techniques"> <div class="models"> <p><span>Models: </span>Homo sapiens</p> </div> </div> <div class="snippet_bottom-left"> <div class="views-citations"> <span class="views"> Downloads: <span class="green-text">162</span> </span> </div> <div class="version-video"> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4140-8_16</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4140-8_16" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4140-8_16" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4140-8_16/MediaObjects/532738_2_En_16_Fig1_HTML.png" alt="Neurobiotin-filled parasol-type retinal ganglion cellsRetinal ganglion cells (RGCs) in the NHP model of glaucoma."> </a> <a href="/articles/10.1007/978-1-0716-4140-8_16" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4140-8_16" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4140-8_16/MediaObjects/532738_2_En_16_Fig2_HTML.jpg" alt="Parasol retinal ganglion cellRetinal ganglion cell (RGC) (human) labelled more than 20 h postmortem by intracellular filling with Lucifer Yellow."> </a> <a href="/articles/10.1007/978-1-0716-4140-8_16" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4140-8_16" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4140-8_16/MediaObjects/532738_2_En_16_Fig3_HTML.png" alt="Retinal ganglion cellsRetinal ganglion cells (RGCs) labelled by the retrograde transport of Horse Radish Peroxidase (HRP) following direct implantation into the optic nerveOptic nerves (retrobulbar)."> </a> <a href="/articles/10.1007/978-1-0716-4140-8_16" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4140-8_16" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4140-8_16/MediaObjects/532738_2_En_16_Fig4_HTML.png" alt="Sholl plots summarizing dendritic integrity in alpha-type RGCsRetinal ganglion cells (RGCs) in mice with induced ocular hypertensionOcular hypertension (OHT) ."> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4140-8_16" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4140-8_16" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4140-8_16/MediaObjects/532738_2_En_16_Fig1_HTML.png" alt="Neurobiotin-filled parasol-type retinal ganglion cellsRetinal ganglion cells (RGCs) in the NHP model of glaucoma."> </a> <a href="/articles/10.1007/978-1-0716-4140-8_16" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4140-8_16" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4140-8_16/MediaObjects/532738_2_En_16_Fig2_HTML.jpg" alt="Parasol retinal ganglion cellRetinal ganglion cell (RGC) (human) labelled more than 20 h postmortem by intracellular filling with Lucifer Yellow."> </a> <a href="/articles/10.1007/978-1-0716-4140-8_16" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4140-8_16" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4140-8_16/MediaObjects/532738_2_En_16_Fig3_HTML.png" alt="Retinal ganglion cellsRetinal ganglion cells (RGCs) labelled by the retrograde transport of Horse Radish Peroxidase (HRP) following direct implantation into the optic nerveOptic nerves (retrobulbar)."> </a> </div> </div> </li> <li class="SN_search-Results__searched_content" data-track-component="search-result"> <div class="SN_search-Results__searched_content_list"> <div class="snippet_source"> <div class="snippet_top-right"><span class="content-type">Protocol</span></div> <div class="snippet_top-left"> <p> <span class="source italic">Springer Protocols</span> <time datetime="" class="date-time"> (2025) </time> </p> </div> </div> <div class="snippet_source_details"> <span><b> Series: </b></span> <span class="italics">Methods In Molecular Biology</span> <span class="separator"><b> > </b></span> <span class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4200-9"><span class="italics">Psychoneuroimmunology</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4200-9_8, Springer Protocols, 2' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4200-9_8'>Hair and Saliva Endocannabinoid and Steroid Hormone Analysis by Liquid Chromatography Paired with Tandem Mass Spectrometry</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">Madeline Jarvis </span> <span class="comma_space">, </span> <span class="author-name">Khalisa Amir Hamzah </span> <span class="comma_space">, </span> <span class="author-name">David Nichols </span> <span class="comma_space">, </span> <span class="author-name">Luke J. Ney <a class="author-email" href="mailto:luke.ney@qut.edu.au"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>Endocannabinoids are lipid neurotransmitters that play an important part in human health. Recent methods have found that quantification of endocannabinoids in hair and saliva samples is possible using liquid chromatography paired with tandem mass</p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4200-9_8'>more</a> </span> </div> <div class="snippet-details hidden"><p>Endocannabinoids are lipid neurotransmitters that play an important part in human health. Recent methods have found that quantification of endocannabinoids in hair and saliva samples is possible using liquid chromatography paired with tandem mass spectrometry (LC-MS/MS). This chapter describes two simple sample preparation methods that can be used to prepare hair and saliva samples for analysis using LC-MS/MS. Our LC-MS/MS method can be applied to both hair and saliva samples and is sufficiently sensitive for endocannabinoid, as well as steroid hormone, quantification in both of these sample matrices. This chapter provides a comprehensive description of how this can be achieved and provides tips and tricks for troubleshooting problems users may experience.</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p class="collapsed"> <span>Techniques: </span>Mass Spectrometry, Liquid Chromatography (LC), Tandem Mass Spectrometry, Liquid Chromatography-tandem Mass Spectrometry, Protein Precipitation... <a class="more-link" href="#">3 more</a> </p> <p class="expanded hidden"> <span>Techniques: </span>Mass Spectrometry, Liquid Chromatography (LC), Tandem Mass Spectrometry, Liquid Chromatography-tandem Mass Spectrometry, Protein Precipitation, Multiple Reaction Monitoring (MRM), Positive Ion Mode Electrospray Ionization, Liquid Chromatography-mass Spectrometry <a class="less-link" href="#">less</a> </p> </div> </div> <div class="snippet_bottom-left"> <div class="version-video"> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4200-9_8</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4200-9_8" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4200-9_8" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4200-9_8/MediaObjects/605386_3_En_8_Fig1_HTML.png" alt="Labeling procedure for an example participant (participant code: MAOL01) who provided three saliva samples and a baseline saliva sample during the experiment"> </a> <a href="/articles/10.1007/978-1-0716-4200-9_8" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4200-9_8" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4200-9_8/MediaObjects/605386_3_En_8_Fig2_HTML.png" alt="Example of chromatography of this method for cortisol, progesterone, arachidonoyl ethanolamide (AEA), 2-arachidonoyl glycerol (2-AG) and its isomer 1-AG, and oleoylethanolamide (OEA)"> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4200-9_8" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4200-9_8" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4200-9_8/MediaObjects/605386_3_En_8_Fig1_HTML.png" alt="Labeling procedure for an example participant (participant code: MAOL01) who provided three saliva samples and a baseline saliva sample during the experiment"> </a> <a href="/articles/10.1007/978-1-0716-4200-9_8" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4200-9_8" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4200-9_8/MediaObjects/605386_3_En_8_Fig2_HTML.png" alt="Example of chromatography of this method for cortisol, progesterone, arachidonoyl ethanolamide (AEA), 2-arachidonoyl glycerol (2-AG) and its isomer 1-AG, and oleoylethanolamide (OEA)"> </a> </div> </div> </li> <li class="SN_search-Results__searched_content" data-track-component="search-result"> <div class="SN_search-Results__searched_content_list"> <div class="snippet_source"> <div class="snippet_top-right"><span class="content-type">Protocol</span></div> <div class="snippet_top-left"> <p> <span class="source italic">Springer Protocols</span> <time datetime="" class="date-time"> (2025) </time> </p> </div> </div> <div class="snippet_source_details"> <span><b> Series: </b></span> <span class="italics">Methods In Molecular Biology</span> <span class="separator"><b> > </b></span> <span class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4220-7"><span class="italics">Golden Gate Cloning</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4220-7_2, Springer Protocols, 3' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4220-7_2'>Golden Gate Cloning of Multigene Constructs Using the Modular Cloning System MoClo</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">Sylvestre Marillonnet <a class="author-email" href="mailto:smarillo@ipb-halle.de"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> <span class="comma_space">, </span> <span class="author-name">Stefan Werner </span> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for construct engineering for biological research and synthetic biology. These systems are simple to use, efficient, and allow users to assemble multigene</p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4220-7_2'>more</a> </span> </div> <div class="snippet-details hidden"><p>Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for construct engineering for biological research and synthetic biology. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the modular cloning system MoClo. Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p class="collapsed"> <span>Techniques: </span>Construct Engineering, DNA Ligation, Microbial Culture, DNA Extraction, Plasmid Purification... <a class="more-link" href="#">2 more</a> </p> <p class="expanded hidden"> <span>Techniques: </span>Construct Engineering, DNA Ligation, Microbial Culture, DNA Extraction, Plasmid Purification, Gel Electrophoresis, Snapgene <a class="less-link" href="#">less</a> </p> </div> </div> <div class="snippet-techniques"> <div class="models"> <p><span>Models: </span>Saccharomyces cerevisiae</p> </div> </div> <div class="snippet_bottom-left"> <div class="views-citations"> <span class="views"> Downloads: <span class="green-text">362</span> </span> </div> <div class="version-video"> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4220-7_2</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4220-7_2" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_2" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_2/MediaObjects/601704_1_En_2_Fig1_HTML.png" alt="Comparison of multigene assembly using level 2 versus levels M and P."> </a> <a href="/articles/10.1007/978-1-0716-4220-7_2" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_2" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_2/MediaObjects/601704_1_En_2_Fig2_HTML.png" alt="Level 0 modules (standardStandard parts) and cloning vectors."> </a> <a href="/articles/10.1007/978-1-0716-4220-7_2" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_2" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_2/MediaObjects/601704_1_En_2_Fig3_HTML.png" alt="Level 1 destination vectors."> </a> <a href="/articles/10.1007/978-1-0716-4220-7_2" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_2" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_2/MediaObjects/601704_1_En_2_Fig4_HTML.png" alt="Example for planning a multigene constructMultigene constructs with 18 genes."> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4220-7_2" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_2" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_2/MediaObjects/601704_1_En_2_Fig1_HTML.png" alt="Comparison of multigene assembly using level 2 versus levels M and P."> </a> <a href="/articles/10.1007/978-1-0716-4220-7_2" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_2" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_2/MediaObjects/601704_1_En_2_Fig2_HTML.png" alt="Level 0 modules (standardStandard parts) and cloning vectors."> </a> <a href="/articles/10.1007/978-1-0716-4220-7_2" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_2" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_2/MediaObjects/601704_1_En_2_Fig3_HTML.png" alt="Level 1 destination vectors."> </a> </div> </div> </li> <li class="SN_search-Results__searched_content" data-track-component="search-result"> <div class="SN_search-Results__searched_content_list"> <div class="snippet_source"> <div class="snippet_top-right"><span class="content-type">Protocol</span></div> <div class="snippet_top-left"> <p> <span class="source italic">Springer Protocols</span> <time datetime="" class="date-time"> (2025) </time> </p> </div> </div> <div class="snippet_source_details"> <span><b> Series: </b></span> <span class="italics">Methods In Molecular Biology</span> <span class="separator"><b> > </b></span> <span class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4220-7"><span class="italics">Golden Gate Cloning</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4220-7_14, Springer Protocols, 4' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4220-7_14'>Generating Site Saturation Mutagenesis Libraries and Transferring Them to Broad Host-Range Plasmids Using Golden Gate Cloning</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">Niels N. Oehlmann </span> <span class="comma_space">, </span> <span class="author-name">Johannes G. Rebelein <a class="author-email" href="mailto:johannes.rebelein@mpi-marburg.mpg.de"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a</p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4220-7_14'>more</a> </span> </div> <div class="snippet-details hidden"><p>Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (<span class="EmphasisTypeItalic">anfHDGK</span>) of <span class="EmphasisTypeItalic">Rhodobacter capsulatus,</span> which is subsequently screened for the improved formation of molecular hydrogen.</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p class="collapsed"> <span>Techniques: </span>Mutagenesis, Agarose Gel Electrophoresis, Protein Engineering, Touchdown PCR, DNA Purification... <a class="more-link" href="#">8 more</a> </p> <p class="expanded hidden"> <span>Techniques: </span>Mutagenesis, Agarose Gel Electrophoresis, Protein Engineering, Touchdown PCR, DNA Purification, DNA Ligation, DNA Extraction, Sanger Sequencing, Gel Imaging, In Vivo Assay, Recombinant Protein Expression, Gas Chromatography (GC), PCR <a class="less-link" href="#">less</a> </p> </div> </div> <div class="snippet-techniques"> <div class="models"> <p><span>Models: </span>Escherichia coli, Saccharomyces cerevisiae</p> </div> </div> <div class="snippet_bottom-left"> <div class="views-citations"> <span class="views"> Downloads: <span class="green-text">359</span> </span> </div> <div class="version-video"> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4220-7_14</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4220-7_14" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_14" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_14/MediaObjects/601704_1_En_14_Fig1_HTML.jpg" alt="Workflow for the saturation mutagenesis library generationLibrary generation of the Fe nitrogenaseIron nitrogenase gene anfD in the broad host range plasmidBroad-host range plasmid pMM0128."> </a> <a href="/articles/10.1007/978-1-0716-4220-7_14" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_14" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_14/MediaObjects/601704_1_En_14_Fig2_HTML.png" alt="(a) Workflow of the in vivo screening of anfD library in R."> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4220-7_14" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_14" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_14/MediaObjects/601704_1_En_14_Fig1_HTML.jpg" alt="Workflow for the saturation mutagenesis library generationLibrary generation of the Fe nitrogenaseIron nitrogenase gene anfD in the broad host range plasmidBroad-host range plasmid pMM0128."> </a> <a href="/articles/10.1007/978-1-0716-4220-7_14" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4220-7_14" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4220-7_14/MediaObjects/601704_1_En_14_Fig2_HTML.png" alt="(a) Workflow of the in vivo screening of anfD library in R."> </a> </div> </div> </li> <li class="SN_search-Results__searched_content" data-track-component="search-result"> <div class="SN_search-Results__searched_content_list"> <div class="snippet_source"> <div class="snippet_top-right"><span class="content-type">Protocol</span></div> <div class="snippet_top-left"> <p> <span class="source italic">Springer Protocols</span> <time datetime="" class="date-time"> (2025) </time> </p> </div> </div> <div class="snippet_source_details"> <span><b> Series: </b></span> <span class="italics">Methods In Molecular Biology</span> <span class="separator"><b> > </b></span> <span class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4136-1"><span class="italics">Computational Methods for 3D Genome Analysis</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4136-1_21, Springer Protocols, 5' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4136-1_21'>Learning Enhancer–Gene associations from Bulk Transcriptomic and Epigenetic Sequencing Data with STITCHIT</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">Laura Rumpf </span> <span class="comma_space">, </span> <span class="author-name">Marcel H. Schulz <a class="author-email" href="mailto:marcel.schulz@em.uni-frankfurt.de"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>To reveal gene regulation mechanisms, it is essential to understand the role of regulatory elements, which are possibly distant from gene promoters. Integrative analysis of epigenetic and transcriptomic data can be used to gain insights into</p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4136-1_21'>more</a> </span> </div> <div class="snippet-details hidden"><p>To reveal gene regulation mechanisms, it is essential to understand the role of regulatory elements, which are possibly distant from gene promoters. Integrative analysis of epigenetic and transcriptomic data can be used to gain insights into gene-expression regulation in specific phenotypes. Here, we discuss STITCHIT, an approach to dissect epigenetic variation in a gene-specific manner across many samples for the identification of regulatory elements without relying on peak calling algorithms. The obtained genomic regions are then further refined using a regularized linear model approach, which can also be used to predict gene expression. We illustrate the use of STITCHIT using H3k27ac ChIP-seq and RNA-seq data from the International Human Epigenome Consortium (IHEC).</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p><span>Techniques: </span>Peak Calling, ChIP-seq, RNA-seq, Ensembl</p> </div> </div> <div class="snippet-techniques"> <div class="models"> <p><span>Models: </span>Homo sapiens</p> </div> </div> <div class="snippet_bottom-left"> <div class="views-citations"> <span class="views"> Downloads: <span class="green-text">384</span> </span> </div> <div class="version-video"> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4136-1_21</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4136-1_21" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4136-1_21" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4136-1_21/MediaObjects/605906_1_En_21_Fig1_HTML.png" alt="Overview of all tissues and cell types and their frequency in the IHEC dataset, for which RNA-seq and H3K27ac ChIP-seq were measured"> </a> <a href="/articles/10.1007/978-1-0716-4136-1_21" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4136-1_21" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4136-1_21/MediaObjects/605906_1_En_21_Fig2_HTML.png" alt="Output plot generated by the discretization approach for IHEC sample IHECRE00000001 on chromosome 21 genes (Subheading 2)."> </a> <a href="/articles/10.1007/978-1-0716-4136-1_21" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4136-1_21" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4136-1_21/MediaObjects/605906_1_En_21_Fig3_HTML.png" alt="Overview of the STITCHIT algorithm."> </a> <a href="/articles/10.1007/978-1-0716-4136-1_21" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4136-1_21" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4136-1_21/MediaObjects/605906_1_En_21_Fig4_HTML.png" alt="Performance assessment in sixfold outer Cross-Validation procedure between predicted and measured gene expressions."> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4136-1_21" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4136-1_21" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4136-1_21/MediaObjects/605906_1_En_21_Fig1_HTML.png" alt="Overview of all tissues and cell types and their frequency in the IHEC dataset, for which RNA-seq and H3K27ac ChIP-seq were measured"> </a> <a href="/articles/10.1007/978-1-0716-4136-1_21" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4136-1_21" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4136-1_21/MediaObjects/605906_1_En_21_Fig2_HTML.png" alt="Output plot generated by the discretization approach for IHEC sample IHECRE00000001 on chromosome 21 genes (Subheading 2)."> </a> <a href="/articles/10.1007/978-1-0716-4136-1_21" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4136-1_21" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4136-1_21/MediaObjects/605906_1_En_21_Fig3_HTML.png" alt="Overview of the STITCHIT algorithm."> </a> </div> </div> </li> <li class="SN_search-Results__searched_content" data-track-component="search-result"> <div class="SN_search-Results__searched_content_list"> <div class="snippet_source"> <div class="snippet_top-right"><span class="content-type">Protocol</span></div> <div class="snippet_top-left"> <p> <span class="source italic">Springer Protocols</span> <time datetime="" class="date-time"> (2025) </time> </p> </div> </div> <div class="snippet_source_details"> <span><b> Series: </b></span> <span class="italics">Methods In Molecular Biology</span> <span class="separator"><b> > </b></span> <span class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4128-6"><span class="italics">Immunosenescence</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4128-6_13, Springer Protocols, 6' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4128-6_13'>In Vitro Interaction Between Yeast Extracellular Vesicles and Human Monocyte-Derived Dendritic Cells</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">Roberta Amoriello <a class="author-email" href="mailto:roberta.amoriello@unifi.it"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> <span class="comma_space">, </span> <span class="author-name">Stefano Nenciarini </span> <span class="comma_space">, </span> <span class="author-name">Duccio Cavalieri </span> <span class="comma_space">, </span> <span class="author-name">Clara Ballerini </span> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and</p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4128-6_13'>more</a> </span> </div> <div class="snippet-details hidden"><p>Extracellular vesicles (EVs) are lipid-bound particles produced by a wide variety of cells from different biological species. EVs can carry molecules, such as nucleic acids and metabolites, and are involved in cell functioning, communication, and signaling. Recent literature reported that pathogenic or commensal yeast strains can produce EVs targeting the host’s immune system and exerting immunomodulatory actions. In humans, yeast EVs can be endocytosed by dendritic cells (DCs), characterized by phagocyting and migrating capabilities with the role of capturing antigens to present to T lymphocytes, triggering the immune response. Physiological or disease-associated immunosenescence impairs both DC functionality and gut microbiota; thus investigating the interaction between commensal microorganisms and the host’s immune system would help elucidate the impact of aging on the immune system-microbiota interplay. We hereby present a protocol for the incubation of in vitro<span class="EmphasisTypeItalic">-</span>generated human monocyte-derived DCs with EVs purified from different yeast strains isolated from fermented milk. The protocol includes flow cytometry analysis on DC activation markers and endocytosis assay.</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p class="collapsed"> <span>Techniques: </span>Immunomodulation, Flow Cytometry, Ultrafiltration, Ultracentrifugation, Data analysis... <a class="more-link" href="#">2 more</a> </p> <p class="expanded hidden"> <span>Techniques: </span>Immunomodulation, Flow Cytometry, Ultrafiltration, Ultracentrifugation, Data analysis, Cell And Tissue Culture, Cell Separation And Isolation <a class="less-link" href="#">less</a> </p> </div> </div> <div class="snippet-techniques"> <div class="models"> <p><span>Models: </span>Saccharomyces cerevisiae, Homo sapiens</p> </div> </div> <div class="snippet-techniques"> <div class="antibodies"> <p><span>Antibodies: </span>Anti-human, Anti-CD14, Anti-CD11c</p> </div> </div> <div class="snippet_bottom-left"> <div class="views-citations"> <span class="views"> Downloads: <span class="green-text">287</span> </span> </div> <div class="version-video"> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4128-6_13</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4128-6_13" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4128-6_13" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4128-6_13/MediaObjects/539463_1_En_13_Fig1_HTML.png" alt="Experiment of endocytosisEndocytosis by moDCs of yeast EVs."> </a> <a href="/articles/10.1007/978-1-0716-4128-6_13" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4128-6_13" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4128-6_13/MediaObjects/539463_1_En_13_Fig2_HTML.png" alt="Gating strategyGating strategies for flow cytometryFlow cytometry analysis on moDC activation markers."> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4128-6_13" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4128-6_13" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4128-6_13/MediaObjects/539463_1_En_13_Fig1_HTML.png" alt="Experiment of endocytosisEndocytosis by moDCs of yeast EVs."> </a> <a href="/articles/10.1007/978-1-0716-4128-6_13" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4128-6_13" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4128-6_13/MediaObjects/539463_1_En_13_Fig2_HTML.png" alt="Gating strategyGating strategies for flow cytometryFlow cytometry analysis on moDC activation markers."> </a> </div> </div> </li> <li class="SN_search-Results__searched_content" data-track-component="search-result"> <div class="SN_search-Results__searched_content_list"> <div class="snippet_source"> <div class="snippet_top-right"><span class="content-type">Protocol</span></div> <div class="snippet_top-left"> <p> <span class="source italic">Springer Protocols</span> <time datetime="" class="date-time"> (2025) </time> </p> </div> </div> <div class="snippet_source_details"> <span><b> Series: </b></span> <span class="italics">Methods and Protocols in Food Science</span> <span class="separator"><b> > </b></span> <span class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4144-6"><span class="italics">Dairy Foods Processing</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4144-6_19, Springer Protocols, 7' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4144-6_19'>Ice Cream and Frozen Yogurt</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">Eliara Acipreste Hudson </span> <span class="comma_space">, </span> <span class="author-name">Letícia Bruni Souza </span> <span class="comma_space">, </span> <span class="author-name">Jaqueline de Paula Rezende </span> <span class="comma_space">, </span> <span class="author-name">Ana Clarissa dos Santos Pires <a class="author-email" href="mailto:ana.pires@ufv.br"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>Frozen desserts are emulsion-based products that undergo the freezing process while being aerated and are consumed in their frozen state. Notably, the popularity of frozen desserts has seen a remarkable increase in recent years. Among the various</p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4144-6_19'>more</a> </span> </div> <div class="snippet-details hidden"><p>Frozen desserts are emulsion-based products that undergo the freezing process while being aerated and are consumed in their frozen state. Notably, the popularity of frozen desserts has seen a remarkable increase in recent years. Among the various frozen desserts available, ice cream and frozen yogurt are prominent choices. Ice cream is a complex multiphase system composed of ice crystals, air cells, and fat globules embedded in a highly viscous, freeze-concentrated matrix phase. The production of frozen yogurt closely resembles that of ice cream, with the key distinction in the fermentation of the mix using yogurt cultures before freezing. This fermentation imparts distinctive flavors and textures to the final product. The ingredients used in ice cream and frozen yogurt production can vary to achieve the desired physical, chemical, and sensory properties. High-quality ingredients are crucial for maintaining the quality and flavor of these products. The production of these frozen desserts involves several critical steps, each playing a crucial role in determining the ultimate product’s texture, structure, flavor, and overall quality. This chapter provides a comprehensive guide to the processing of ice cream and frozen yogurt, encompassing essential steps, technical considerations, and practical insights. It is tailored for food scientists, technologists, and professionals engaged in producing frozen desserts.</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p><span>Techniques: </span>Fermentation, Homogenization</p> </div> </div> <div class="snippet_bottom-left"> <div class="views-citations"> <span class="views"> Downloads: <span class="green-text">11</span> </span> </div> <div class="version-video"> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4144-6_19</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4144-6_19" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4144-6_19" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4144-6_19/MediaObjects/607980_1_En_19_Fig1_HTML.png" alt="Flowchart with ice cream manufacturing steps"> </a> <a href="/articles/10.1007/978-1-0716-4144-6_19" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4144-6_19" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4144-6_19/MediaObjects/607980_1_En_19_Fig2_HTML.png" alt="Frozen yogurt preparation flowchart"> </a> <a href="/articles/10.1007/978-1-0716-4144-6_19" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4144-6_19" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4144-6_19/MediaObjects/607980_1_En_19_Fig3_HTML.png" alt="(a) Schematic of a scraped surface heat exchanger (SSHE) used in ice cream manufacturing."> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4144-6_19" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4144-6_19" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4144-6_19/MediaObjects/607980_1_En_19_Fig1_HTML.png" alt="Flowchart with ice cream manufacturing steps"> </a> <a href="/articles/10.1007/978-1-0716-4144-6_19" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4144-6_19" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4144-6_19/MediaObjects/607980_1_En_19_Fig2_HTML.png" alt="Frozen yogurt preparation flowchart"> </a> <a href="/articles/10.1007/978-1-0716-4144-6_19" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4144-6_19" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4144-6_19/MediaObjects/607980_1_En_19_Fig3_HTML.png" alt="(a) Schematic of a scraped surface heat exchanger (SSHE) used in ice cream manufacturing."> </a> </div> </div> </li> <li class="SN_search-Results__searched_content" data-track-component="search-result"> <div class="SN_search-Results__searched_content_list"> <div class="snippet_source"> <div class="snippet_top-right"><span class="content-type">Protocol</span></div> <div class="snippet_top-left"> <p> <span class="source italic">Springer Protocols</span> <time datetime="" class="date-time"> (2025) </time> </p> </div> </div> <div class="snippet_source_details"> <span><b> Series: </b></span> <span class="italics">Methods In Molecular Biology</span> <span class="separator"><b> > </b></span> <span class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4168-2"><span class="italics">Schizosaccharomyces pombe</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4168-2_12, Springer Protocols, 8' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4168-2_12'>Proliferation Analyses of Conditional Knockdown Strains Using CRISPR Interference in Fission Yeast</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">Ken Ishikawa <a class="author-email" href="mailto:ishikawa_ken@kurume-u.ac.jp"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> <span class="comma_space">, </span> <span class="author-name">Shigeaki Saitoh </span> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>CRISPR interference is a method to conditionally inhibit transcription of an arbitrary target gene. This is useful to study the functions of essential genes, which are required for cellular viability. Although many conditional gene perturbation</p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4168-2_12'>more</a> </span> </div> <div class="snippet-details hidden"><p>CRISPR interference is a method to conditionally inhibit transcription of an arbitrary target gene. This is useful to study the functions of essential genes, which are required for cellular viability. Although many conditional gene perturbation techniques are available for <span class="EmphasisTypeItalic">Schizosaccharomyces pombe</span>, CRISPRi facilitates construction of a large number of knockdown strains because of its systematic, simple procedure. Here, we describe a method to construct and characterize knockdown strains using dCas9-mediated CRISPRi in <span class="EmphasisTypeItalic">S. pombe</span>, including a variation of CRISPRi induction technique in a 96-well format for high-throughput studies.</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p class="collapsed"> <span>Techniques: </span>CRISPRi, Clonogenic Assay, DNA Ligation, DNA Extraction, Cell And Tissue Culture... <a class="more-link" href="#">3 more</a> </p> <p class="expanded hidden"> <span>Techniques: </span>CRISPRi, Clonogenic Assay, DNA Ligation, DNA Extraction, Cell And Tissue Culture, DNA Purification, Restriction Enzyme Digestion, Agarose Gel Electrophoresis <a class="less-link" href="#">less</a> </p> </div> </div> <div class="snippet-techniques"> <div class="models"> <p><span>Models: </span>Schizosaccharomyces pombe, Escherichia coli, Saccharomyces cerevisiae</p> </div> </div> <div class="snippet_bottom-left"> <div class="version-video"> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4168-2_12</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4168-2_12" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_12" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_12/MediaObjects/606823_1_En_12_Fig1_HTML.png" alt="Structure of the pSPdCas9 plasmid."> </a> <a href="/articles/10.1007/978-1-0716-4168-2_12" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_12" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_12/MediaObjects/606823_1_En_12_Fig2_HTML.png" alt="PCRPCR primers for plasmid structure confirmation."> </a> <a href="/articles/10.1007/978-1-0716-4168-2_12" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_12" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_12/MediaObjects/606823_1_En_12_Fig3_HTML.png" alt="Assembly of a 96-well filter plate and a micro-vacuum manifold."> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4168-2_12" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_12" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_12/MediaObjects/606823_1_En_12_Fig1_HTML.png" alt="Structure of the pSPdCas9 plasmid."> </a> <a href="/articles/10.1007/978-1-0716-4168-2_12" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_12" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_12/MediaObjects/606823_1_En_12_Fig2_HTML.png" alt="PCRPCR primers for plasmid structure confirmation."> </a> <a href="/articles/10.1007/978-1-0716-4168-2_12" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_12" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_12/MediaObjects/606823_1_En_12_Fig3_HTML.png" alt="Assembly of a 96-well filter plate and a micro-vacuum manifold."> </a> </div> </div> </li> <li class="SN_search-Results__searched_content" data-track-component="search-result"> <div class="SN_search-Results__searched_content_list"> <div class="snippet_source"> <div class="snippet_top-right"><span class="content-type">Protocol</span></div> <div class="snippet_top-left"> <p> <span class="source italic">Springer Protocols</span> <time datetime="" class="date-time"> (2025) </time> </p> </div> </div> <div class="snippet_source_details"> <span><b> Series: </b></span> <span class="italics">Methods In Molecular Biology</span> <span class="separator"><b> > </b></span> <span class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4168-2"><span class="italics">Schizosaccharomyces pombe</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4168-2_5, Springer Protocols, 9' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4168-2_5'>Live Imaging of Fission Yeast Single-Cell Lineages Using a Microfluidic Device</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">Hidenori Nakaoka <a class="author-email" href="mailto:nakaoka.hidenori@tokushima-u.ac.jp"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>Mother machine (MM) is a microfluidic device originally developed for long-term live imaging of <span class="EmphasisTypeItalic">Escherichia coli</span> bacterial cells under a microscope. The simple yet sophisticated design has enabled microbiologists to track multiple single-cell</p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4168-2_5'>more</a> </span> </div> <div class="snippet-details hidden"><p>Mother machine (MM) is a microfluidic device originally developed for long-term live imaging of <span class="EmphasisTypeItalic">Escherichia coli</span> bacterial cells under a microscope. The simple yet sophisticated design has enabled microbiologists to track multiple single-cell lineages cultured under highly controlled external environments. Here, I describe how to fabricate a fission yeast version of MM with photolithography and soft lithography. Procedures for setting up the microfluidic device for long-term live microscopy are also explained.</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p class="collapsed"> <span>Techniques: </span>Photolithography, Soft Lithography, Live Cell Imaging, Microscopy, Vacuum Deposition... <a class="more-link" href="#">4 more</a> </p> <p class="expanded hidden"> <span>Techniques: </span>Photolithography, Soft Lithography, Live Cell Imaging, Microscopy, Vacuum Deposition, Cross-linking, Biopsy, Replica Molding, Degassing <a class="less-link" href="#">less</a> </p> </div> </div> <div class="snippet-techniques"> <div class="models"> <p><span>Models: </span>Schizosaccharomyces pombe</p> </div> </div> <div class="snippet_bottom-left"> <div class="version-video"> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4168-2_5</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4168-2_5" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_5" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_5/MediaObjects/606823_1_En_5_Fig1_HTML.png" alt="Making photomasks."> </a> <a href="/articles/10.1007/978-1-0716-4168-2_5" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_5" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_5/MediaObjects/606823_1_En_5_Fig2_HTML.png" alt="Making alignment marks on a silicon wafer."> </a> <a href="/articles/10.1007/978-1-0716-4168-2_5" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_5" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_5/MediaObjects/606823_1_En_5_Fig3_HTML.png" alt="Making SU-8 molds on a silicon wafer."> </a> <a href="/articles/10.1007/978-1-0716-4168-2_5" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_5" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_5/MediaObjects/606823_1_En_5_Fig4_HTML.png" alt="Making an epoxy replica mold."> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4168-2_5" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_5" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_5/MediaObjects/606823_1_En_5_Fig1_HTML.png" alt="Making photomasks."> </a> <a href="/articles/10.1007/978-1-0716-4168-2_5" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_5" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_5/MediaObjects/606823_1_En_5_Fig2_HTML.png" alt="Making alignment marks on a silicon wafer."> </a> <a href="/articles/10.1007/978-1-0716-4168-2_5" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4168-2_5" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4168-2_5/MediaObjects/606823_1_En_5_Fig3_HTML.png" alt="Making SU-8 molds on a silicon wafer."> </a> </div> </div> </li> <li class="SN_search-Results__searched_content" data-track-component="search-result"> <div class="SN_search-Results__searched_content_list"> <div class="snippet_source"> <div class="snippet_top-right"><span class="content-type">Protocol</span></div> <div class="snippet_top-left"> <p> <span class="source italic">Springer Protocols</span> <time datetime="" class="date-time"> (2025) </time> </p> </div> </div> <div class="snippet_source_details"> <span><b> Series: </b></span> <span class="italics">Methods In Molecular Biology</span> <span class="separator"><b> > </b></span> <span class="springer-book-title"> <span><b>Book: </b></span> <a target="_blank" rel="noopener" href="https://dx.doi.org/10.1007/978-1-0716-4264-1"><span class="italics">Mitochondrial Bioenergetics</span></a> </span> </div> <h3> <a class="title" target="_blank" rel="noopener" data-track="click" data-track-action='searchresult_interact' data-track-label='10.1007/978-1-0716-4264-1_7, Springer Protocols, 10' data-track-category='mosaic:search-page' href='/articles/10.1007/978-1-0716-4264-1_7'>Mitochondrial Membrane Potential (ΔΨ) Fluctuations Associated with the Metabolic States of Mitochondria</a> </h3> <div class="author-info"> <span class="authors"> <span class="author-name">Ivo F. Machado </span> <span class="comma_space">, </span> <span class="author-name">Raul G. Miranda </span> <span class="comma_space">, </span> <span class="author-name">João S. Teodoro </span> <span class="comma_space">, </span> <span class="author-name">Daniel J. Dorta </span> <span>... </span><span class="author-name">Carlos M. Palmeira </span> <a class="author-email" href="mailto:palmeira@ci.uc.pt"> <img class="author-email-icon" src="/assets/images/icons/email.png" alt="Email to Author"/> </a> </span> </div> <div class="snippet"> <div> <div class="trimmedText"><p>The proton electrochemical gradient generated by the respiratory chain activity accounts for over 90% of the available respiratory energy and, as such, its evaluation and accurate measurement regarding total values and fluctuations are an invaluable </p>  <span class="read-more">… <a href="#" class="more-link" data-track="click" data-track-action="snippet_interact" data-track-category="mosaic:search-page" data-track-label='10.1007/978-1-0716-4264-1_7'>more</a> </span> </div> <div class="snippet-details hidden"><p>The proton electrochemical gradient generated by the respiratory chain activity accounts for over 90% of the available respiratory energy and, as such, its evaluation and accurate measurement regarding total values and fluctuations are an invaluable component of the understanding of mitochondrial function. Consequently, alterations in electric potential across the inner mitochondrial membrane generated by differential protonic accumulation and transport are known as the mitochondrial membrane potential, or Δψ, and are reflective of the functional metabolic status of mitochondria. There are several experimental approaches to measure Δψ, ranging from fluorometric evaluations to electrochemical probes. In this chapter, we describe how Δψ may be evaluated in isolated mitochondria and live cells using electrochemical and fluorescent methods, such as tetraphenylphosphonium (TPP+) and tetramethylrhodamine methyl ester (TMRM), respectively. These methods are dependent on the accumulation of cationic probes within mitochondria, which are assessed by using a TPP<sup>+</sup>-selective electrode or instruments that measure fluorescence (microplate reader and flow cytometer).</p> <span class="read-less"> <a href="#" class="less-link">less</a></span> </div> </div> </div> <div class="snippet-techniques"> <div class="techniques"> <p class="collapsed"> <span>Techniques: </span>Flow Cytometry, Cervical Dislocation, Decapitation, Magnetic Stirring, Cell Staining... <a class="more-link" href="#">1 more</a> </p> <p class="expanded hidden"> <span>Techniques: </span>Flow Cytometry, Cervical Dislocation, Decapitation, Magnetic Stirring, Cell Staining, Cell And Tissue Culture <a class="less-link" href="#">less</a> </p> </div> </div> <div class="snippet-techniques"> <div class="models"> <p><span>Models: </span>Homo sapiens, Bos taurus, CM-2 [Human ESC]</p> </div> </div> <div class="snippet_bottom-left"> <div class="version-video"> <span class="versions">Versions available</span> </div> </div> <div class="p_doi" hidden>10.1007/978-1-0716-4264-1_7</div> <div class="image-search hiddenDesktop"> <a href="/articles/10.1007/978-1-0716-4264-1_7" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4264-1_7" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4264-1_7/MediaObjects/540707_3_En_7_Fig1_HTML.png" alt="Mitochondrial electron transport chainElectron transport chain and dissipation of the proton gradient by uncoupling proteins, converting electrochemical energy into heat."> </a> <a href="/articles/10.1007/978-1-0716-4264-1_7" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4264-1_7" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4264-1_7/MediaObjects/540707_3_En_7_Fig2_HTML.png" alt="Schematic representation of TPP+ distribution across the mitochondrial membrane, according to the Nernst equation"> </a> <a href="/articles/10.1007/978-1-0716-4264-1_7" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4264-1_7" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4264-1_7/MediaObjects/540707_3_En_7_Fig3_HTML.png" alt="Typical traces obtained with a TPP+ electrode."> </a> </div> <div class="image-search hiddenMobile"> <a href="/articles/10.1007/978-1-0716-4264-1_7" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4264-1_7" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4264-1_7/MediaObjects/540707_3_En_7_Fig1_HTML.png" alt="Mitochondrial electron transport chainElectron transport chain and dissipation of the proton gradient by uncoupling proteins, converting electrochemical energy into heat."> </a> <a href="/articles/10.1007/978-1-0716-4264-1_7" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4264-1_7" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4264-1_7/MediaObjects/540707_3_En_7_Fig2_HTML.png" alt="Schematic representation of TPP+ distribution across the mitochondrial membrane, according to the Nernst equation"> </a> <a href="/articles/10.1007/978-1-0716-4264-1_7" data-track-category="mosaic:search-page" data-track="click" data-track-action="snippetimage_interact" data-track-label="10.1007/978-1-0716-4264-1_7" target="_blank"> <img src="https://media.springernature.com/w200/springer-static/image/chp%3A10.1007%2F978-1-0716-4264-1_7/MediaObjects/540707_3_En_7_Fig3_HTML.png" alt="Typical traces obtained with a TPP+ electrode."> </a> </div> </div> </li> </ul> <div class="pagination_bottom" data-track-component="pagination"> <div class="pagination-bar"> <div class="pagination-bar-form" id="pagination-form" autocomplete="off"> <span class="prev span-prev"></span> <span class="page-nr-wrapper"> Page <input id="pageNo" name="Page no" title="Page no" class="page-number" value=1 type="number" min="1" max="7749" data-track="click" data-track-category="mosaic:search-page" data-track-action="pagination_interact" data-track-label='1' required pattern="\d*" aria-label="page-number"> <input id="pageNumber" class="InputText" type="hidden" name="pageNumber" value=1 /> of <span id="totalNumberOfPages" class="pagination__number-of-pages">7749</span> </span> <a 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