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Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System. 使用 SpyCatcher-SpyTag 系统对外膜蛋白质进行荧光标记。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3734-0_4
Rachael Duodu, Dirk Linke, Jack C Leo
{"title":"Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System.","authors":"Rachael Duodu, Dirk Linke, Jack C Leo","doi":"10.1007/978-1-0716-3734-0_4","DOIUrl":"10.1007/978-1-0716-3734-0_4","url":null,"abstract":"<p><p>The SpyCatcher-SpyTag system has become a popular and versatile tool for protein ligation. It is based on a small globular protein (SpyCatcher) that binds to a 13-residue peptide (SpyTag), which subsequently leads to the formation of a covalent isopeptide bond. Thus, the reaction is essentially irreversible. Here, we describe how the SpyCatcher-SpyTag system can be used to label surface-exposed bacterial outer membrane proteins, e.g., for topology mapping or fluorescent time-course experiments. We cover using fluorescence measurements and microscopy to measure labeling efficiency using SpyCatcher fused with superfolder GFP in this chapter.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2778 ","pages":"53-63"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119959","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Monitoring the Interaction of the Peptidoglycan with the Bacterial β-Barrel Assembly Machinery. 监测肽聚糖与细菌 β 管组装机制的相互作用
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3734-0_11
Federico Corona, Waldemar Vollmer
{"title":"Monitoring the Interaction of the Peptidoglycan with the Bacterial β-Barrel Assembly Machinery.","authors":"Federico Corona, Waldemar Vollmer","doi":"10.1007/978-1-0716-3734-0_11","DOIUrl":"10.1007/978-1-0716-3734-0_11","url":null,"abstract":"<p><p>Gram-negative bacteria coordinate the biosynthesis of their different cell envelope components. Growth of the outer membrane (OM) requires the essential β-barrel assembly machine (BAM), which inserts OM proteins (OMPs) into the OM. The underlying peptidoglycan (PG) sacculus grows by the insertion of nascent glycan chains. We have previously identified interactions between BAM and PG in E. coli and showed that these interactions coordinate OM biogenesis with PG growth. BAM responds to the maturation state of the PG, and this mechanism activates preferentially BAM complexes at sites of active PG synthesis. Here we present protocols to purify soluble Bam proteins and full-length BamABCDE, isolate PG and soluble PG fragments, and study BAM-PG interactions with the isolated components. We also describe the protocol to detect interactions between Bam proteins and PG in cells.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2778 ","pages":"159-183"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119964","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
In Vitro Tumorigenic Assay: A Tumor Sphere Assay for Cancer Stem Cells. 体外致癌测定：癌症干细胞的肿瘤球试验。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_7
Amani Yehya, Hisham Bahmad, Wassim Abou-Kheir
{"title":"In Vitro Tumorigenic Assay: A Tumor Sphere Assay for Cancer Stem Cells.","authors":"Amani Yehya, Hisham Bahmad, Wassim Abou-Kheir","doi":"10.1007/978-1-0716-3730-2_7","DOIUrl":"10.1007/978-1-0716-3730-2_7","url":null,"abstract":"<p><p>Cancer stem cells (CSCs) represent a subpopulation of tumor cells that are thought to be responsible for therapy resistance, recurrence, and metastasis through their capacity to self-renew and differentiate into heterogeneous downstream lineages of cancer cells. Understanding the features of CSCs is crucial for managing cancer disease and establishing potential targeted therapeutics. Tumor sphere formation assay is a widely used in vitro method that selects and enriches the CSC subpopulation from the total population of cancer cells, based on their inherent ability to grow and clonally expand in serum-free and nonadherent culture conditions. Here we provide a detailed methodology to generate and propagate spheres from isolated cell suspensions of tumor tissues and cell lines using a semisolid Matrigel<sup>TM</sup>-based three-dimensional (3D) culture system.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2777 ","pages":"91-98"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Methods to Study the Role of Mechanical Signals in the Induction of Cancer Stem Cells. 研究机械信号在诱导癌症干细胞中的作用的方法。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3730-2_13
Alessandro Gandin, Paolo Contessotto, Tito Panciera
{"title":"Methods to Study the Role of Mechanical Signals in the Induction of Cancer Stem Cells.","authors":"Alessandro Gandin, Paolo Contessotto, Tito Panciera","doi":"10.1007/978-1-0716-3730-2_13","DOIUrl":"10.1007/978-1-0716-3730-2_13","url":null,"abstract":"<p><p>Microenvironmental mechanical signals are fundamental regulators of cell behavior both in physiological and in pathological context, particularly in the induction and maintenance of tumorigenic properties. It is thus of utmost importance to experimentally recreate conditions that mimic the physical attributes of real tissues to study their impact on cell behavior and in particular on the induction of cancer stem cell (CSC) properties. Here we present protocols to investigate the role of mechanical stiffness on reprogramming of primary mammary gland cells into CSCs, including the synthesis of hydrogel substrates of the desired stiffness, the isolation and culture of primary differentiated normal cells derived from the human mammary gland, and the assessment of their CSC attributes after oncogene-mediated transformation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2777 ","pages":"177-189"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Mechanical Stimulation of Adipose-Derived Stromal/Stem Cells for Functional Tissue Engineering of the Musculoskeletal System via Cyclic Hydrostatic Pressure, Simulated Microgravity, and Cyclic Tensile Strain. 通过循环静水压、模拟微重力和循环拉伸应变对脂肪基质/干细胞进行机械刺激，以实现肌肉骨骼系统的功能性组织工程学。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3762-3_25
Rachel C Nordberg, Josie C Bodle, Elizabeth G Loboa
{"title":"Mechanical Stimulation of Adipose-Derived Stromal/Stem Cells for Functional Tissue Engineering of the Musculoskeletal System via Cyclic Hydrostatic Pressure, Simulated Microgravity, and Cyclic Tensile Strain.","authors":"Rachel C Nordberg, Josie C Bodle, Elizabeth G Loboa","doi":"10.1007/978-1-0716-3762-3_25","DOIUrl":"10.1007/978-1-0716-3762-3_25","url":null,"abstract":"<p><p>It is critical that human adipose-derived stromal/stem cell (hASC) tissue engineering therapies possess appropriate mechanical properties in order to restore the function of the load-bearing tissues of the musculoskeletal system. In an effort to elucidate hASC response to mechanical stimulation and develop mechanically robust tissue-engineered constructs, recent research has utilized a variety of mechanical loading paradigms, including cyclic tensile strain, cyclic hydrostatic pressure, and mechanical unloading in simulated microgravity. This chapter will describe the methods for applying these mechanical stimuli to hASC to direct differentiation for functional tissue engineering of the musculoskeletal system.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2783 ","pages":"349-365"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140119997","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Repurposing Decellularized Lung to Generate Vascularized Fat. 重新利用脱细胞肺来生成血管化脂肪。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3762-3_18
Lindsey K Huff, Zihan Ling, Megan K DeBari, Xi Ren, Rosalyn D Abbott
{"title":"Repurposing Decellularized Lung to Generate Vascularized Fat.","authors":"Lindsey K Huff, Zihan Ling, Megan K DeBari, Xi Ren, Rosalyn D Abbott","doi":"10.1007/978-1-0716-3762-3_18","DOIUrl":"10.1007/978-1-0716-3762-3_18","url":null,"abstract":"<p><p>Conventional therapies to address critically sized defects in subcutaneous adipose tissue remain a reconstructive challenge for surgeons, largely due to the lack of graft pre-vascularization. Adipose tissue relies on a dense microvasculature network to deliver nutrients, oxygen, nonadipose tissue-derived growth factors, cytokines, and hormones, as well as transporting adipose tissue-derived endocrine signals to other organ systems. This chapter addresses these vascularization issues by combining decellularized lung matrices with a step-wise seeding of patient-specific adipose-derived stem cells and endothelial cells to develop large-volume, perfusable, and pre-vascularized adipose grafts.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2783 ","pages":"269-278"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140120002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial β-Barrel Assembly Machine. 通过特定位点光交联研究细菌β-管状组装机介导的毒素传输
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3734-0_8
Emily M Bouzan, Christine L Hagan
{"title":"Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial β-Barrel Assembly Machine.","authors":"Emily M Bouzan, Christine L Hagan","doi":"10.1007/978-1-0716-3734-0_8","DOIUrl":"10.1007/978-1-0716-3734-0_8","url":null,"abstract":"<p><p>Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative organisms that relies on a specific interaction between a CdiA protein on the surface of one cell and a β-barrel protein on the surface of a neighboring cell. This interaction triggers the transport of a protein toxin into the neighboring cell where it exerts its lethal activity. Several classes of CdiA proteins that bind to different β-barrel receptors have been identified, but the molecular mechanism by which they deliver their toxins across the outer membranes of their target cells is poorly understood. Here we describe the use of site-specific photocrosslinking to characterize the interaction between a CdiA protein and its receptor. We describe the method for an E. coli CdiA that utilizes BamA as its receptor. BamA's central role in assembling β-barrel proteins in the outer membrane makes its role in CDI particularly intriguing; it suggests that these two different protein transport processes might share mechanistic features. Our in vitro photocrosslinking method is useful in elucidating early steps in the CDI mechanism, but it could be adapted to study later steps or to study other CdiA-receptor pairs.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2778 ","pages":"117-131"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140120013","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Canine Adult Adipose Tissue-Derived Multipotent Stromal Cell Isolation, Characterization, and Differentiation. 犬成体脂肪组织衍生多能基质细胞的分离、表征和分化。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3762-3_8
Takashi Taguchi, Mandi J Lopez
{"title":"Canine Adult Adipose Tissue-Derived Multipotent Stromal Cell Isolation, Characterization, and Differentiation.","authors":"Takashi Taguchi, Mandi J Lopez","doi":"10.1007/978-1-0716-3762-3_8","DOIUrl":"10.1007/978-1-0716-3762-3_8","url":null,"abstract":"<p><p>Adult mesenchymal stromal/stem cells (MSCs) are a standard component of de novo tissue generation to treat and study injury, disease, and degeneration. Canine patients constitute a major component of veterinary practice, and dogs share numerous pathologic conditions with humans. The relative abundance of adipose-derived stromal/stem cells (ASCs) in various canine adipose tissue depots is well described. Refined isolation, characterization, and differentiation techniques contribute to the collective knowledge of ASC phenotypes and subpopulations for specific tissue targets. Continued efforts to advance the knowledge of canine ASC behavior in vivo are critical to harnessing the full potential of primary cell isolates. This chapter contains a description of techniques to isolate, characterize, and differentiate canine ASCs.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2783 ","pages":"115-136"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140120023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
In Vitro Culture of White Adipose Tissue. 白色脂肪组织的体外培养
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3762-3_19
Jake J Fontenot, Frank H Lau
{"title":"In Vitro Culture of White Adipose Tissue.","authors":"Jake J Fontenot, Frank H Lau","doi":"10.1007/978-1-0716-3762-3_19","DOIUrl":"10.1007/978-1-0716-3762-3_19","url":null,"abstract":"<p><p>White adipose tissue (WAT) plays a crucial endocrine organ that regulates blood glucose and lipid levels, satiety, and inflammation. Before the described technique, primary white adipocytes could not be stably cultured in vitro. The lack of a reliable primary culture model impeded research in WAT metabolism and drug development. We have developed a novel technique for WAT primary culture called \"sandwiched white adipose tissue\" (SWAT). SWAT overcomes the natural buoyancy of adipocytes by sandwiching minced WAT between sheets of adipose-derived stromal cells. The resulting constructs are viable for at least 8 weeks in culture. SWAT maintains the intact extracellular matrix, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2783 ","pages":"279-285"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140120028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting. 通过 Western Blotting 验证 ELISpot 抗体特异性的当前趋势。
Methods in molecular biology Pub Date : 2024-01-01 DOI: 10.1007/978-1-0716-3690-9_2
Biji T Kurien, R Hal Scofield
{"title":"Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting.","authors":"Biji T Kurien, R Hal Scofield","doi":"10.1007/978-1-0716-3690-9_2","DOIUrl":"https://doi.org/10.1007/978-1-0716-3690-9_2","url":null,"abstract":"<p><p>The enzyme-linked immunospot (ELISpot) assay is a highly useful and sensitive method to detect total immunoglobulin and antigen-specific antibody-secreting cells. In addition, this method can measure biological activity and immunological secretions from immune cells. In general, membrane-bound antigen allows binding of antibody secreted by B cells, or a membrane-bound analyte-specific antibody binds to the specific analyte (e.g., cytokines) elicited from cells added to the well containing the bound antibody. The response from added cells is then detected by using an anti-Ig antibody and a colorimetric substrate, while in the case of non-B cells, the elicited antigen is detected with appropriate antibodies and enzyme-conjugated antibodies. Specificity of antibodies binding the protein of interest is necessary to achieve correct results. Western blotting can be used for this with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. Despite its general simplicity, western blotting is a powerful technique for immunodetection of proteins (notably low abundance proteins) as it provides simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Now, we have plethora of immunoblotting methods to validate antibodies for ELISpot.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2768 ","pages":"15-27"},"PeriodicalIF":0.0,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140175428","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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