Methods in molecular biology最新文献_第5页

Spheroid Invasion Assay of Melanoma Cells by Hanging Drop Technique. 挂滴法检测黑素瘤细胞球形浸润。

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_615

Asiye Busra Boz Er, Ceren Sumer

引用次数: 0

Establishment of 3D Tooth Organoid Culture from Early-Postnatal Mouse Molar and Incisor. 生后早期小鼠磨牙和门牙三维类器官培养的建立。

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_623

Florian Hermans, Hugo Vankelecom, Annelies Bronckaers, Ivo Lambrichts

引用次数: 0

Methodology for Assessing Drug Efficacy: Protocol for Single and Combination Drug Screening Using HeLa Cell Cultures. 评估药物疗效的方法学：使用HeLa细胞培养进行单一和联合药物筛选的方案。

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_605

Phumuzile Dube, Bernice Monchusi, Mutsa M Takundwa, Vanelle L Kenmogne, Austin Malise, Deepak B Thimiri Govinda Raj

{"title":"Methodology for Assessing Drug Efficacy: Protocol for Single and Combination Drug Screening Using HeLa Cell Cultures.","authors":"Phumuzile Dube, Bernice Monchusi, Mutsa M Takundwa, Vanelle L Kenmogne, Austin Malise, Deepak B Thimiri Govinda Raj","doi":"10.1007/7651_2025_605","DOIUrl":"https://doi.org/10.1007/7651_2025_605","url":null,"abstract":"This protocol outlines a detailed method for performing drug sensitivity testing (DST) on HeLa cells, focusing on both single-drug and combination-drug screening to assess cell viability. DST is integral to cancer research and functional precision medicine, providing insight into individual drug responses and facilitating the optimization of drug combinations. The protocol includes preparing and maintaining HeLa cell cultures, seeding in 96-well plates, and performing single and combination drug treatments using a low-throughput screening approach. These drug treatments aim to evaluate therapeutic effectiveness, enhance understanding of synergistic interactions, and identify optimal combinations that could minimize toxicity while overcoming resistance. Data analysis uses open-source tools, including the BREEZE pipeline and Synergy-Finder, allowing for precise analysis of cell viability and drug interactions. This protocol provides a robust, reproducible framework for DST in cancer research, applicable to other cell lines, patient samples, and various drug types/classes. The critical role of DST in improving clinical treatment strategies through precise, scalable drug response analysis is demonstrated.","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":"143663736","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

Deep Learning-Driven Computational Approaches for Studying Intrinsically Disordered Regions in S100-A9. 基于深度学习驱动的S100-A9内部无序区域研究方法

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_617

Gionathan L Distefano, Fabio D'Amico

引用次数: 0

Application of Cerium Oxide Nanozymes (CeONZs) in Human Pluripotent Stem Cell-Derived Cardiomyocytes. 氧化铈纳米酶（CeONZs）在人多能干细胞衍生心肌细胞中的应用。

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_606

Chengwen Hang, Huixin Guo, Mohamed S Moawad, Christie M Sayes, Yi-Han Chen, Jian Yang

{"title":"Application of Cerium Oxide Nanozymes (CeONZs) in Human Pluripotent Stem Cell-Derived Cardiomyocytes.","authors":"Chengwen Hang, Huixin Guo, Mohamed S Moawad, Christie M Sayes, Yi-Han Chen, Jian Yang","doi":"10.1007/7651_2025_606","DOIUrl":"https://doi.org/10.1007/7651_2025_606","url":null,"abstract":"Cardiovascular diseases (CVDs) are a leading cause of death globally. Excessive production of reactive oxygen species (ROS) is detrimental to cardiomyocytes (CMs), triggering inflammation, inducing cell death, disrupting calcium homeostasis, and leading to arrhythmia. Thus, ROS is considered a common pathological factor in CVDs. Although the efficacy of antioxidants targeting ROS is currently limited, nanotechnology offers opportunities to develop antioxidants with improved selectivity and bioavailability, which can effectively prevent or treat oxidative stress-related CVDs. Cerium oxide nanozymes (CeONZs) can efficiently scavenge excessive ROS by mimicking the activity of endogenous antioxidant enzymes. However, their nanosafety and efficacy in human CMs remain unclear, posing a critical issue to be addressed before clinical applications. Due to the scarcity of primary human CMs, human pluripotent stem cells (hPSCs) and their derived cardiomyocytes (hPSC-CMs) provide a valuable source for modeling CVDs and their therapeutic interventions. This chapter presents a preparation method for CeONZs and outlines the assessment of their biosafety and antioxidant efficacy in hPSC-CMs.","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":"143662481","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

Co-colonization and Co-culture of Lung Alveoli Epithelial Stem Cells and Their Endothelial Niche Cells. 肺泡上皮干细胞及其内皮小生境细胞的共定殖和共培养。

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_607

Hiroyuki Hirai, Qian Chen, Yuru Liu

{"title":"Co-colonization and Co-culture of Lung Alveoli Epithelial Stem Cells and Their Endothelial Niche Cells.","authors":"Hiroyuki Hirai, Qian Chen, Yuru Liu","doi":"10.1007/7651_2025_607","DOIUrl":"10.1007/7651_2025_607","url":null,"abstract":"The maintenance of lung function relies crucially on the homeostatic replacement and post-injury regeneration of the lung alveolar epithelium (Hogan et al., Cell Stem Cell 15(2):123-138, 2014). Dysfunctions in these processes contribute to the pathogenesis of numerous lung diseases (Hogan et al., Cell Stem Cell 15(2):123-138, 2014; Matthay et al., J Clin Invest 122(8):2731-2740, 2012). While identifying stem and progenitor cells in the lung epithelium has significantly enriched our understanding of endogenous replacement and regenerative mechanisms (Hogan et al., Cell Stem Cell 15(2):123-138, 2014), it is clear that epithelial cells interact closely with mesenchymal components which create a micro-environmental niche that is vital for regulating both homeostatic replacement and post-injury regeneration of epithelial cells. Specific subsets of alveolar type II cells (AT2) behave as epithelial stem cells of the distal lung. We have identified a CD44high subpopulation of AT2 cells that are preferentially located near macro-blood vessels and manifest stem cell characteristics (Chen et al., Stem Cell Rep 19(6):890-905, 2024; Am J Physiol Lung Cell Mol Physiol 313(1):L41-l51, 2017). In addition, the macro-blood vessels endothelial cells (ECs) function as niche components to support the CD44high AT2s. Here, we describe the method to identify the CD44high AT2 cells by immuno-fluorescence and co-culture of CD44high AT2 cells with lung endothelial cells-their potential niche component-in 3D organoid culture.","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":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12279409/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143662713","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Protocol Development for CRISPR/Cas9 Knockout of the Anti-inflammatory Protein TNIP1 in HaCaT Keratinocytes. CRISPR/Cas9敲除HaCaT角质形成细胞中抗炎蛋白TNIP1的方案开发

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_616

Liam E Carman, Michael L Samulevich, Brian J Aneskievich

{"title":"Protocol Development for CRISPR/Cas9 Knockout of the Anti-inflammatory Protein TNIP1 in HaCaT Keratinocytes.","authors":"Liam E Carman, Michael L Samulevich, Brian J Aneskievich","doi":"10.1007/7651_2025_616","DOIUrl":"https://doi.org/10.1007/7651_2025_616","url":null,"abstract":"Gene editing in cultured cells, including the intent of sequence disruption to achieve a functional knockout of the targeted gene, has been greatly facilitated with the advent of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) technology. Primary cell strains and immortalized cell lines from diverse tissue types have been successfully targeted both for basic research examining the effects of loss of the correlating protein and for modeling select loss-of-function disorders. Such accomplishments have extended to cutaneous cells, especially epidermal keratinocytes given their key structural and functional role in barrier formation and surveillance of and response to surface events such as triggering and processing inflammatory responses. Here we describe disruption of the Tumor Necrosis factor-induced protein 3-Interacting Protein 1 (TNIP1) gene in human HaCaT keratinocytes to generate an ongoing loss of expression as a parallel system to transient knockdown we had previously achieved with siRNA transfection. The TNIP1 protein restricts cytoplasmic progression of inflammatory signals. We cover our CRISPR/Cas9 vector choice, enrichment of transfected cells via positive selection for puromycin resistance, their subsequent cloning, and gene disruption and expression analysis. We also emphasize prior keratinocyte-CRISPR/Cas9 literature as a springboard for other investigators and to illustrate the widespread relevance of such editing to the diverse, yet highly consequentially different, genes expressed in keratinocytes.","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":"143663738","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

Generation of Functional Brain Region-Specific Neural Spheroids for High Throughput Screening. 用于高通量筛选的功能性脑区域特异性神经球体的生成。

Methods in molecular biology Pub Date : 2025-02-19 DOI: 10.1007/7651_2024_593

Jiajing Zhang, Angelica Medina, Marc Ferrer, Emily M Lee

{"title":"Generation of Functional Brain Region-Specific Neural Spheroids for High Throughput Screening.","authors":"Jiajing Zhang, Angelica Medina, Marc Ferrer, Emily M Lee","doi":"10.1007/7651_2024_593","DOIUrl":"https://doi.org/10.1007/7651_2024_593","url":null,"abstract":"Therapeutic development and research in the neurodegenerative disease field encounters many challenges such as availability of reproducible and scalable cellular model systems that are biologically, physiologically, and pharmacologically relevant. These cellular models must be informative of cellular mechanisms of diseases and predictive for therapeutics efficacy and toxicity testing during drug discovery and development. Neural spheroids fill the gap of cellular models of the brain that are functional, versatile in neural cell type composition, robust, and scalable for high-throughput screening (HTS). We have previously developed a protocol to aggregate pre-determined ratios of differentiated human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes in a scaffold-free environment to form 3D brain-region specific spheroids. By mixing different neuronal types, neural spheroids can be used to simulate the neuronal-type heterogeneity of distinct brain regions in vivo, including the prefrontal cortex (PFC) and ventral tegmental area (VTA). Here, we present a detailed description of a method for generating functional brain region-specific spheroids with HTS-compatible assay readout that monitors changes in neural network activity by measuring calcium oscillations. The versatility of the platform is such that these neural spheroids cellular assays are applicable for a wide range of disease modeling, compound validation, and screening and are limited only by the availability of input cells, including neural subtype, disease cells, and immune cells such as microglia.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143441335","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

Generation of Retinal Organoids Using Human-Induced Pluripotent Stem Cells. 利用人诱导的多能干细胞生成视网膜类器官。

Methods in molecular biology Pub Date : 2025-02-11 DOI: 10.1007/7651_2024_600

Keith Theodore, Joel Alan Imventarza, Saleha Tahir, Bruna Lopes da Costa, Peter M J Quinn

引用次数: 0

Generation and Characterization of a New Aging Skin Human Dermal Extracellular Matrix Scaffold. 一种新型老化皮肤人真皮细胞外基质支架的制备与表征。

Methods in molecular biology Pub Date : 2025-01-18 DOI: 10.1007/7651_2024_579

Estibaliz Fernández-Carro, Jesús Ciriza

引用次数: 0