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Bimolecular Fluorescence Complementation (BiFC) Technique for Exocytic Proteins in Murine Hippocampal Neurons. 小鼠海马神经元胞外蛋白的双分子荧光互补技术。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_20
Gözdem Karapinar Kapucu, Thorsten Trimbuch, Christian Rosenmund, Marion Weber-Boyvat
{"title":"Bimolecular Fluorescence Complementation (BiFC) Technique for Exocytic Proteins in Murine Hippocampal Neurons.","authors":"Gözdem Karapinar Kapucu, Thorsten Trimbuch, Christian Rosenmund, Marion Weber-Boyvat","doi":"10.1007/978-1-0716-4314-3_20","DOIUrl":"10.1007/978-1-0716-4314-3_20","url":null,"abstract":"<p><p>The bimolecular fluorescence complementation (BiFC) technique is a powerful tool for visualizing protein-protein interactions in vivo. It involves genetically fused nonfluorescent fragments of green fluorescent protein (GFP) or its variants to the target proteins of interest. When these proteins interact, the GFP fragments come together, resulting in the reconstitution of a functional fluorescent protein complex that can be observed using fluorescence microscopy. In this chapter, we provide a detailed overview of the BiFC method and its application in studying protein-protein interactions in mouse hippocampal neurons. We discuss experimental procedures, including virus construct design, neuronal transduction, and imaging optimization. Additionally, we explore complementary assays for result validation and address potential challenges associated with BiFC experiments in the neuronal system. Overall, the BiFC offers researchers a valuable approach for investigating the spatial and temporal dynamics of protein interactions in living neuronal cells.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"281-294"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979136","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
High-Resolution Imaging of Intracellular Trafficking of B Cell Receptor Using Specific Hybridization Internalization Probe (SHIP). 利用特异性杂交内化探针（SHIP）对B细胞受体细胞内运输进行高分辨率成像。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4442-3_6
Sara Hernández-Pérez, Pieta K Mattila
{"title":"High-Resolution Imaging of Intracellular Trafficking of B Cell Receptor Using Specific Hybridization Internalization Probe (SHIP).","authors":"Sara Hernández-Pérez, Pieta K Mattila","doi":"10.1007/978-1-0716-4442-3_6","DOIUrl":"10.1007/978-1-0716-4442-3_6","url":null,"abstract":"<p><p>Recent advancements in microscopy have greatly expanded our understanding of intracellular traffic. Yet, due to the inherent characteristics of B cells, such as their small size and high receptor density on the plasma membrane, visualization of internalized cargo or receptors remains challenging. This challenge is particularly pronounced in the case of the B cell receptor (BCR), where accurate detection of internalized, antigen-bound BCR molecules can be strongly hindered by the signal from the plasma membrane-bound pool of the same molecules.To tackle this issue, we adapted the Specific Hybridization Internalization Probe (SHIP) assay, initially designed for flow cytometry studies, for the study of BCR internalization using microscopy. This assay utilizes a single-stranded DNA (ssDNA) fluorescence internalization probe (FIP) paired with a complementary ssDNA quenching probe that \"turns off\" the signal from the (extracellular) surface-bound BCRs, greatly facilitating the unambiguous identification of internalized (intracellular) receptors. Moreover, the assay is versatile and adaptable to a range of imaging modalities, including live-cell imaging and super-resolution microscopy. SHIP proves to be a valuable tool in the study of intracellular processes, offering enhanced imaging precision for the detection of internalized BCRs.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2909 ","pages":"73-82"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542645","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
Use of Human Acute Myeloid Leukemia Cells to Study Graft-Versus-Leukemia Immunity in Xenogeneic Mouse Models of Graft-Versus-Host Disease. 利用人急性髓系白血病细胞研究移植物抗宿主病小鼠异种模型的移植物抗白血病免疫
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4430-0_17
Charline Faville, Bianca E Silva, Frédéric Baron, Grégory Ehx
{"title":"Use of Human Acute Myeloid Leukemia Cells to Study Graft-Versus-Leukemia Immunity in Xenogeneic Mouse Models of Graft-Versus-Host Disease.","authors":"Charline Faville, Bianca E Silva, Frédéric Baron, Grégory Ehx","doi":"10.1007/978-1-0716-4430-0_17","DOIUrl":"10.1007/978-1-0716-4430-0_17","url":null,"abstract":"<p><p>Allogeneic hematopoietic cell transplantation (allo-HCT) is the main therapeutic approach for patients with high-risk acute myeloid leukemia (AML), but the rate of relapse remains high and is associated with poor outcomes. Discovering new approaches to maximize the graft-versus-leukemia (GVL) effects while mitigating graft-versus-host disease (GVHD) should therefore be pursued. Because of the difficulties in modeling AML in mice, patient-derived xenotransplantations (PDXs) in immunodeficient NOD-scid-IL2rg<sup>null</sup> (NSG) mice are preferred to study the GVL effects. In PDX, AML is typically induced through the intravenous injection of cell lines or leukemic blasts obtained from patients. GVHD and GVL effects are induced by (co)-injecting human T cells or peripheral blood mononuclear cells (PBMCs). While this approach enables the induction of systemic leukemia, notably developing in the spleen and bone marrow of the animals, it can also be associated with difficulties in monitoring the disease, notably by flow cytometry. This can be circumvented by using luciferase-expressing AML cells or transplanting the leukemic cells in Matrigel to generate solid tumors that are easier to monitor. Here, we provide detailed instructions on how to prepare human PBMCs and leukemic cells, transplant them, and monitor the disease in NSG mice.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2907 ","pages":"359-375"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657771","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
Utilization of Clinical Data and Evaluation of Biomarkers in the Investigation of Graft-Versus-Host Disease Outcomes. 移植物抗宿主病预后研究中临床数据的利用和生物标志物的评估。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4430-0_3
Serhat Çelik, Leylagül Kaynar
{"title":"Utilization of Clinical Data and Evaluation of Biomarkers in the Investigation of Graft-Versus-Host Disease Outcomes.","authors":"Serhat Çelik, Leylagül Kaynar","doi":"10.1007/978-1-0716-4430-0_3","DOIUrl":"10.1007/978-1-0716-4430-0_3","url":null,"abstract":"<p><p>Graft-versus-host disease (GVHD) is one of the most important obstacles after allogeneic hematopoietic stem cell transplantation (allo-HCT). The mortality rate is around 50%, especially in severe GVHD. One of the most important clinical outcomes in GVHD is non-relapse mortality (NRM). NRM was defined as death without evidence of relapse or progression. Kaplan Meier, log-rank test, and Cox model are used in survival analysis methods. There are various biomarkers that assess clinical outcomes of GVHD. Damage-associated molecular patterns, pathogen-associated molecular patterns, microRNAs, markers of endothelial dysfunction, cytokines, and their receptors are used to predict the occurrence of GVHD and clinical outcomes in GVHD. Furthermore, the utilization of panels that assess many biomarkers has proven to be successful in predicting the clinical outcomes of GVHD, particularly NRM.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2907 ","pages":"71-83"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143657784","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
Agroinoculation of Plants with Geminiviruses. 双病毒植物的农接种。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4454-6_5
Anelise F Orílio
{"title":"Agroinoculation of Plants with Geminiviruses.","authors":"Anelise F Orílio","doi":"10.1007/978-1-0716-4454-6_5","DOIUrl":"10.1007/978-1-0716-4454-6_5","url":null,"abstract":"<p><p>Agroinoculation refers to the introduction of genetic information from plant viruses into leaves or other organs of the host plant through Agrobacterium tumefaciens. Agroinoculation of geminiviruses requires the insertion of tandem copies of the genome between the T-DNA borders of an Agrobacterium binary vector. The technique is applicable to most plant species and is limited only by the host range of the virus and of the A. tumefaciens strain. Herbaceous plants can be easily agroinoculated using a needleless syringe. The successful agroinoculation of plants with begomoviruses involves the preparation of A. tumefaciens competent cells, the transformation of A. tumefaciens with the cloned tandem copy of the viral genome, and the agroinoculation procedure itself. This chapter describes a straightforward protocol for the agroinoculation of a cloned infectious begomovirus into plants.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2912 ","pages":"29-34"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143597341","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
Untargeted Metabolic Phenotyping by LC-MS. LC-MS的非靶向代谢表型分析。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4334-1_6
Ian D Wilson, Elizabeth Want
{"title":"Untargeted Metabolic Phenotyping by LC-MS.","authors":"Ian D Wilson, Elizabeth Want","doi":"10.1007/978-1-0716-4334-1_6","DOIUrl":"10.1007/978-1-0716-4334-1_6","url":null,"abstract":"<p><p>Untargeted analysis by LC-MS is a valuable tool for metabolic profiling (metabonomics/metabolomics), and applications of this technology have grown rapidly over the past decade. LC-MS offers advantages of speed, sensitivity, relative ease of sample preparation, and large dynamic range compared to other platforms in this role. However, like any analytical approach, there are still drawbacks and challenges that have to be overcome, some of which are being addressed by advances in both column chemistries and instrumentation. In particular, the combination of LC-MS with ion mobility offers many new possibilities for improved analyte separation, detection, and structural identification. There are many untargeted LC-MS approaches which can be applied to metabolic phenotyping, and these usually need to be optimized for the type of sample, the nature of the study, or the biological question. Some of the main LC-MS approaches for untargeted metabolic phenotyping are described in detail in the following protocol.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2891 ","pages":"109-129"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142984053","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
FRET-Based Sensor for Measuring Adenine Nucleotide Binding to AMPK. 基于fret的测量腺苷核苷酸与AMPK结合的传感器。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4284-9_2
Roland Abi Nahed, Martin Pelosse, Francesco Aulicino, Florine Cottaz, Imre Berger, Uwe Schlattner
{"title":"FRET-Based Sensor for Measuring Adenine Nucleotide Binding to AMPK.","authors":"Roland Abi Nahed, Martin Pelosse, Francesco Aulicino, Florine Cottaz, Imre Berger, Uwe Schlattner","doi":"10.1007/978-1-0716-4284-9_2","DOIUrl":"10.1007/978-1-0716-4284-9_2","url":null,"abstract":"<p><p>AMP-activated protein kinase (AMPK) has evolved to detect a critical increase in cellular AMP/ATP and ADP/ATP concentration ratios as a signal for limiting energy supply. Such energy stress then leads to AMPK activation and downstream events that maintain cellular energy homeostasis. AMPK activation by AMP, ADP, or pharmacological activators involves a conformational switch within the AMPK heterotrimeric complex. We have engineered an AMPK-based sensor, AMPfret, which translates the activating conformational switch into a fluorescence signal, based on increased fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores. Here we describe how this sensor can be used to analyze direct AMPK activation by small molecules in vitro using a fluorimeter, or to estimate changes in the energy state of cells using standard fluorescence or confocal microscopy.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2882 ","pages":"15-45"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483693","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
Quantification of Infectious Rhinovirus A and B Serotypes by Plaque Assay. 用空斑法定量测定传染性鼻病毒A、B血清型。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4410-2_4
Mohsen Tabasi, Haleh Ganjian, Umadevi Sajjan
{"title":"Quantification of Infectious Rhinovirus A and B Serotypes by Plaque Assay.","authors":"Mohsen Tabasi, Haleh Ganjian, Umadevi Sajjan","doi":"10.1007/978-1-0716-4410-2_4","DOIUrl":"10.1007/978-1-0716-4410-2_4","url":null,"abstract":"<p><p>Plaque assay is a quantitative assay that determines the number of infective virions in the viral stock or the infected cells. Plaques are essentially virus-infected cells that produce progeny virus and infect adjacent cells. The cells producing progeny virus die and detach from the dish, leaving an empty space in the confluent monolayer of cells. Unlike other viruses, rhinovirus does not form characteristic round plaques but shows small clear areas of different shapes surrounded by dying cells. Rhinoviruses form plaques only in highly susceptible H1HeLa cells but not in their primary target, airway epithelial cells. The plaque assay to determine the number of infective virions works only for rhinovirus species A and B, but not C, because the latter does not infect H1HeLa cells. Here we describe a method to quantify the infective virions by plaque assay for rhinovirus speciesA and B.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2903 ","pages":"31-38"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143523967","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
Evaluation of Constitutive or Induced Activation State of the VLA-4 Integrin in Human and Murine Samples. hla -4整合素在人、鼠体内本构激活或诱导激活状态的评价。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4442-3_4
Erika Tissino, Antonella Zucchetto, Federico Pozzo, Tanja Nicole Hartmann, Valter Gattei
{"title":"Evaluation of Constitutive or Induced Activation State of the VLA-4 Integrin in Human and Murine Samples.","authors":"Erika Tissino, Antonella Zucchetto, Federico Pozzo, Tanja Nicole Hartmann, Valter Gattei","doi":"10.1007/978-1-0716-4442-3_4","DOIUrl":"10.1007/978-1-0716-4442-3_4","url":null,"abstract":"<p><p>The integrin heterodimer CD49d/CD29 (a.k.a. Very Late Antigen-4, VLA-4) mediates cell-cell and cell-matrix interaction through binding to its specific ligands. VLA-4 can be present on the cell surface at different conformation states that affect the binding affinity for the ligands. In chronic lymphocytic leukemia (CLL), higher VLA-4 levels have been demonstrated to be associated with a worse prognosis both in the chemo-immunotherapy era and in the BCR inhibitor setting, in keeping with the role of VLA-4 as a key molecule favoring CLL cell localization in protective niches of bone marrow and lymph nodes. Here, we describe functional flow cytometry-based methods to assess the activation state of the VLA-4 integrin, applicable in both human and murine settings, as well as in fresh or thawed samples. A specific \"R\" script for analyzing flow cytometry data is also provided.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2909 ","pages":"45-60"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542642","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
Cryopreservation of Small Ruminant Sperm and Detection of Sperm Cryocapacitation. 小型反刍动物精子的低温冷冻和精子冷冻能力检测。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4406-5_12
Patricia Peris-Frau, Silvia Gimeno-Martos
{"title":"Cryopreservation of Small Ruminant Sperm and Detection of Sperm Cryocapacitation.","authors":"Patricia Peris-Frau, Silvia Gimeno-Martos","doi":"10.1007/978-1-0716-4406-5_12","DOIUrl":"10.1007/978-1-0716-4406-5_12","url":null,"abstract":"<p><p>Sperm cryopreservation is an indispensable tool for preserving the genetic and epigenetic material of valuable males or endangered species. Here, we describe the protocols most frequently used for conventional cryopreservation of small ruminant sperm, considering differences between the origin of the sperm sample (epididymal vs. ejaculated sperm) and species. In addition, since one of the major drawbacks of sperm cryopreservation is the reduction of viability and fertility due to capacitation-like changes in a considerable amount of sperm, different methods to detect sperm cryocapacitation in small ruminants are explained.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2897 ","pages":"165-182"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143811757","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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