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Three-Dimensional Analysis of Skeletal Muscle Stem Cell Niche Following Tissue Clearing.
Methods in molecular biology Pub Date : 2025-04-05 DOI: 10.1007/7651_2025_618
Yoko Asakura, Smrithi Karthikeyan, Atsushi Asakura
{"title":"Three-Dimensional Analysis of Skeletal Muscle Stem Cell Niche Following Tissue Clearing.","authors":"Yoko Asakura, Smrithi Karthikeyan, Atsushi Asakura","doi":"10.1007/7651_2025_618","DOIUrl":"https://doi.org/10.1007/7651_2025_618","url":null,"abstract":"<p><p>Skeletal muscle is an intricately structured tissue made up of a complex framework of various cell types. The dynamic spatial and temporal relationships among these cells during both homeostasis and periods of injury contribute to the regenerative abilities of skeletal muscle. Currently, there is a deficiency in quantitative assessment, biological role, and the molecular mechanisms that could elucidate a possible juxtavascular niche for muscle satellite cells, a stem cell population for skeletal muscle regeneration. To fully comprehend the regeneration process by muscle satellite cells, a three-dimensional (3-D) imaging approach is essential. Confocal microscopy serves as an exceptional method for examining the spatial arrangement of cells within a specific tissue. In this protocol, we provide a detailed procedure for preparing optically transparent extensor digitorum longus (EDL) skeletal muscle specimens that are appropriate for confocal microscopy and computational 3-D assessment. We outline the steps for sample preparation, which include perfusion fixation and the tissue clearing process for rodent muscle specimens, as well as guidelines for image capture and computational evaluation featuring sample segmentation and 3-D visualization. This methodology can be utilized to characterize diverse cell types, such as muscle satellite cells and capillary endothelial cells found in rodent skeletal muscle.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780471","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
Human Skeletal Muscle Niche Formation and Analysis with Spatial RNA Sequencing.
Methods in molecular biology Pub Date : 2025-04-05 DOI: 10.1007/7651_2025_619
Ben Clock, Michael Hicks
{"title":"Human Skeletal Muscle Niche Formation and Analysis with Spatial RNA Sequencing.","authors":"Ben Clock, Michael Hicks","doi":"10.1007/7651_2025_619","DOIUrl":"https://doi.org/10.1007/7651_2025_619","url":null,"abstract":"<p><p>Formation of the human skeletal muscle can be achieved through xenotransplant of human stem or progenitor cells into mice. Human cells, such as those derived from human pluripotent stem cells (hPSCs), are dissociated from in vitro culture conditions and injected into immune-compromised mice where human cells must form new myofibers and retain or replace the mouse muscle stem cell pool. Efforts to better understand niche interactions will lead to improved regenerative potential that could ameliorate a broad range of muscle diseases. Spatial RNA sequencing of xenografted tissues allows for precise transcriptomic profiling of human muscle stem and progenitor cells in relation to myofibers and their niche throughout the myogenic differentiation process. Herein, we describe the procedures of obtaining high yields of human xenografted transplants and compare the use of various spatial RNA sequencing platforms to uncover stem cell niche formation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780461","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
Complex Spheroids as an Alternative: In Vivo-Like 3D Model for Investigating the Impact of Stromal Cells on the Radiation Response of Tumors.
Methods in molecular biology Pub Date : 2025-04-03 DOI: 10.1007/7651_2025_614
Hanna Sentek, Diana Klein
{"title":"Complex Spheroids as an Alternative: In Vivo-Like 3D Model for Investigating the Impact of Stromal Cells on the Radiation Response of Tumors.","authors":"Hanna Sentek, Diana Klein","doi":"10.1007/7651_2025_614","DOIUrl":"https://doi.org/10.1007/7651_2025_614","url":null,"abstract":"<p><p>Three-dimensional environments that mimic in vivo microenvironments promote native epithelial cell polarity and differentiation by enabling cell-matrix interactions. When tumor cells were embedded as spherical cell aggregates (spheroids) in such microenvironments, particularly in semi-solid extracellular matrices, further the intimate cell-cell adhesion architecture as well as direct cell-matrix interactions can be efficiently recapitulated, including the presence of nutritional and metabolic gradients. The complexity of these spheroids can be further increased by the use of intercalating stromal cells. Here, we describe a simple and rapid method in which tumor and stromal cells are placed in hanging drop culture to generate homogenous and complex spheroids before embedding them in laminin-/collagen IV-rich basement membrane extracts (Matrigel), which in turn can be used as model system for cancer progression and to evaluate the efficacy of anticancer drugs, especially after radiation treatment.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143764526","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 Adult Mouse Brain Neurogenic Niche Behavior Culturing Adult Mice Brain Slice In Vitro.
Methods in molecular biology Pub Date : 2025-04-03 DOI: 10.1007/7651_2025_611
Maria Isabel Alonso, Sonia Martínez-Páramo, Francisco Lamus, Ángel Gato
{"title":"Evaluation of Adult Mouse Brain Neurogenic Niche Behavior Culturing Adult Mice Brain Slice In Vitro.","authors":"Maria Isabel Alonso, Sonia Martínez-Páramo, Francisco Lamus, Ángel Gato","doi":"10.1007/7651_2025_611","DOIUrl":"https://doi.org/10.1007/7651_2025_611","url":null,"abstract":"<p><p>Adult brain neural precursors carry out their biological activity in specific areas in which they are able to self-renew and differentiate into neurons. This is due to a complex microenvironment of cellular interrelations in which soluble factors from the neighboring cells, vascular structures, and the content of the brain ventricle cavity (cerebrospinal fluid) play a key role. This cellular functional entity, known as the \"neurogenic niche,\" is able to generate new mature neurons, which are functionally integrated into the neuronal circuits of the adult mammal brain. The complexity of neurogenic niche signaling, which include biologically active molecules such as growth factors and morphogens, requires an experimental approach in order to create specific modifications of the biological activity of some of these molecules by means of a model of the active neurogenic niche, allowing an evaluation of neural precursor behavior.Here we describe the adaptation of an in vitro culture technique of adult brain slices with selected coronal sections, involving the two main brain neurogenic niches, the sub-ventricular zone (SVZ), and the hippocampus dentate gyrus, together with their associated sub-ependymal zone (SEZ). We explain certain examples of the experimental approach to modify neurogenic niche soluble signaling, implanting latex microbeads as a carrier for soluble signals. Additionally, we introduce an immune-cytochemical approach involving bromodeoxyuridine detection as a neural precursor cellular lineage tracer in combination with different molecular expressions, as a means of testing progressive states of neural precursor differentiation and neuronal maturation.This system represents a suitable strategy for evaluating the biological role of soluble components of the adult brain neurogenic niche.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143764542","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
Detection of Cellular Senescence on Murine Muscle Tissue Sections by Senescence-Associated β-Galactosidase Staining.
Methods in molecular biology Pub Date : 2025-04-03 DOI: 10.1007/7651_2025_612
Wenxin Zhang, Tom H Cheung
{"title":"Detection of Cellular Senescence on Murine Muscle Tissue Sections by Senescence-Associated β-Galactosidase Staining.","authors":"Wenxin Zhang, Tom H Cheung","doi":"10.1007/7651_2025_612","DOIUrl":"https://doi.org/10.1007/7651_2025_612","url":null,"abstract":"<p><p>Staining for the presence of senescence-associated beta-galactosidase (SA-β-gal) serves as a crucial indicator for cellular senescence. This staining assay is based on the elevated activity of β-galactosidase in senescent cells, which cleaves the X-Gal substrate to produce an insoluble blue product in acidic conditions. This enables X-Gal to be visualized using microscopy. In this study, we describe the identification of SA-β-gal<sup>+</sup> cells within murine muscle tissues.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143764541","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
Oligopaint FISH in Drosophila Testes.
Methods in molecular biology Pub Date : 2025-04-03 DOI: 10.1007/7651_2025_613
Romir Raj, Vedansh Patel, Mayu Inaba
{"title":"Oligopaint FISH in Drosophila Testes.","authors":"Romir Raj, Vedansh Patel, Mayu Inaba","doi":"10.1007/7651_2025_613","DOIUrl":"https://doi.org/10.1007/7651_2025_613","url":null,"abstract":"<p><p>Fluorescence in situ hybridization (FISH) is commonly performed to visualize RNA and DNA. It is routinely used in cytogenetics and karyotyping to check for chromosomal abnormalities and diagnose and identify diseases (Levsky and Singer, J Cell Sci 116:2833-2838, 2003). Oligopaints is a recently established DNA FISH method that has quickly become popular because of its flexibility and economical durability. Oligopaints uses computationally designed PCR-renewable oligonucleotides for probes, which can cover a few kilobases to whole chromosomes (Beliveau, Proc Natl Acad Sci 109:21301-21306, 2012). In addition, different fluorophores can be used for desired probe sets for multicolor imaging. Here, we describe an optimized method for implementing the Oligopaints procedure to visualize genomic regions in Drosophila testis. We further discuss the possibility of resolving local microstructure of specific gene loci.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143764543","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
Testicular Organoid Formation in Microwell Culture.
Methods in molecular biology Pub Date : 2025-03-28 DOI: 10.1007/7651_2025_624
Nathalia de Lima E Martins Lara, Anja Elsenhans, Ina Dobrinski
{"title":"Testicular Organoid Formation in Microwell Culture.","authors":"Nathalia de Lima E Martins Lara, Anja Elsenhans, Ina Dobrinski","doi":"10.1007/7651_2025_624","DOIUrl":"https://doi.org/10.1007/7651_2025_624","url":null,"abstract":"<p><p>Testicular organoids present an exciting 3D in vitro platform to bridge the gap between 2D culture and animal models in male reproduction research, allowing studies on testicular cell-cell interactions, morphogenesis, development, and the spermatogonial stem cell microenvironment in conditions that are more physiologically relevant. Therefore, research with testicular organoids offers opportunities for fertility preservation, disease modeling, and high throughput reproductive toxicity screening. Our laboratory has developed a simple and reproducible protocol using microwell plates, which facilitate the aggregation of single cells and promote the generation of thousands of homogenous organoids that recapitulate testicular cytoarchitecture and functions. In this protocol, a testicular cell suspension is obtained by enzymatic digestion of immature testes and centrifuged into pyramid-shaped microwells, where cells will aggregate and form organoids after a few days in culture. Here we detail our standard protocol for the generation of porcine testicular organoids, which can also be applied to other mammalian species.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730742","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
Organoid-Immune Cell Co-culture for Stable Live Imaging.
Methods in molecular biology Pub Date : 2025-03-28 DOI: 10.1007/7651_2025_627
Nathalia Ferreira, Frauke Alves, Andrea Markus
{"title":"Organoid-Immune Cell Co-culture for Stable Live Imaging.","authors":"Nathalia Ferreira, Frauke Alves, Andrea Markus","doi":"10.1007/7651_2025_627","DOIUrl":"https://doi.org/10.1007/7651_2025_627","url":null,"abstract":"<p><p>Patient-derived organoids (PDOs) have emerged as a promising model for personalized drug testing. Generated from human tumor samples, PDOs effectively recapitulate the genetic and phenotypic heterogeneity of patient tumors, making them an ideal ex vivo platform for studying therapeutic responses, particularly to chemotherapies. However, their lack of components of the immune system limits their use in immunotherapy testing. The following protocol facilitates the co-culture of PDOs from tumor tissue with HLA-matched peripheral blood mononuclear cells (PBMCs) in a fixed Z-plane for stable live-cell imaging. This three-dimensional co-culture method represents a significant advancement in enabling real-time assessment of immunotherapeutic effects on tumor-derived PDOs by live cell imaging.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730741","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
Efficient Isolation and Ex Vivo Differentiation of Murine Satellite Cells from Healthy and Dystrophic Muscle. 从健康和萎缩性肌肉中高效分离和体外分化小鼠卫星细胞
Methods in molecular biology Pub Date : 2025-03-22 DOI: 10.1007/7651_2025_608
Alessio A Cusmano, Cordell A VanGenderen, Tim O Lorenz, Yafen Yang, Natasha C Chang
{"title":"Efficient Isolation and Ex Vivo Differentiation of Murine Satellite Cells from Healthy and Dystrophic Muscle.","authors":"Alessio A Cusmano, Cordell A VanGenderen, Tim O Lorenz, Yafen Yang, Natasha C Chang","doi":"10.1007/7651_2025_608","DOIUrl":"https://doi.org/10.1007/7651_2025_608","url":null,"abstract":"<p><p>Satellite cells are the stem cells of adult skeletal muscles and confer skeletal muscle with remarkable regenerative ability. Under homeostatic conditions, satellite cells reside in a quiescent state in their niche along the basal lamina of muscle fibers. Upon receiving stimuli, satellite cells activate and engage in regenerative myogenesis to repair damaged fibers. Due to the impact of satellite cell differentiation on muscle physiology, studying their differentiation is relevant both within the context of healthy and diseased muscle. Due to the abundance of cell populations within skeletal muscle, the study of satellite cells is predicated on isolating highly pure populations. Fluorescence activated cell sorting (FACS) represents the gold standard for deriving highly pure satellite cell isolates but is costly and can reduce cell viability. In addition, proliferating satellite cells in vitro invariably transition to a homogeneous myoblast population that bestows a selective advantage on fast-dividing cells, reducing satellite cell heterogeneity. In this chapter, we describe our protocol for magnetic-activated cell sorting (MACS) of satellite cells. MACS preserves cell viability to a greater degree than FACS, and our approach allows for highly pure sorted populations of satellite cells. In addition, sorted cells can enter and progress through the myogenic program immediately upon plating, avoiding the need for lengthy expansion periods.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143674220","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 Anti-aging Agents Using the D-Galactose-Induced Accelerated Aging Model.
Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_609
Serdar Bora Bayraktaroğlu, Raife Dilek Turan, Neslihan Pakize Taşlı, Fikrettin Şahin
{"title":"Evaluation of Anti-aging Agents Using the D-Galactose-Induced Accelerated Aging Model.","authors":"Serdar Bora Bayraktaroğlu, Raife Dilek Turan, Neslihan Pakize Taşlı, Fikrettin Şahin","doi":"10.1007/7651_2025_609","DOIUrl":"https://doi.org/10.1007/7651_2025_609","url":null,"abstract":"<p><p>The aging population is rapidly increasing, emphasizing the importance of understanding aging mechanisms and developing effective anti-aging therapies. This chapter investigates the efficacy of novel anti-aging agents, including exosomes and boron compounds, using the D-galactose-induced accelerated aging model. Both in vitro (skin organoid models) and in vivo (rat models) systems are employed to explore cellular, molecular, and histological changes. This comprehensive analysis provides critical insights into the potential of these agents in reversing age-associated pathologies.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143663720","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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