Bio-protocol最新文献_第8页

Coupled Enzyme Assay for Measuring Ornithine Decarboxylase Activity in Cell Lysates Using a Liquid-Stable CO₂ Detection Reagent. 用液体稳定的CO2检测试剂测定细胞裂解物中鸟氨酸脱羧酶活性的偶联酶试验。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5768

Jung-Mao Hsu

{"title":"Coupled Enzyme Assay for Measuring Ornithine Decarboxylase Activity in Cell Lysates Using a Liquid-Stable CO2 Detection Reagent.","authors":"Jung-Mao Hsu","doi":"10.21769/BioProtoc.5768","DOIUrl":"10.21769/BioProtoc.5768","url":null,"abstract":"Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine biosynthesis that plays a critical role in cell proliferation and tumorigenesis. Reliable quantification of ODC activity is essential for mechanistic and therapeutic studies. Traditional assays often rely on radiolabeled substrates or discontinuous endpoint measurements. Here, we describe a non-radioactive, continuous spectrophotometric assay for measuring ODC activity in cell lysates using a commercially available liquid-stable CO2 detection reagent. In this assay, CO2 generated by ODC is captured as bicarbonate and utilized in a coupled enzymatic system containing phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH), leading to oxidation of thio-NADH. The decrease in absorbance at 405 nm due to thio-NADH oxidation is monitored in real time and is proportional to ODC activity. The protocol is performed in a 96-well plate format, requires minimal reagent preparation, and is suitable for medium- to high-throughput applications. Key features • Non-radioactive, continuous assay for measuring ODC activity. • Utilizes a commercially available liquid-stable CO2 detection reagent, requiring minimal preparation and enabling improved reproducibility. • Real-time monitoring at 405 nm using a standard microplate reader. • Adaptable to a high-throughput 96-well format.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5768"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13461751/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148726509","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

A Novel Plate Reader-Based Protocol for Measurement of DNAJB6 Dimerization Activity. 一种新的基于平板阅读器的DNAJB6二聚化活性测定方法。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5776

Anna Gelman, Leif Kofoed Nielsen, Christian Hansen

{"title":"A Novel Plate Reader-Based Protocol for Measurement of DNAJB6 Dimerization Activity.","authors":"Anna Gelman, Leif Kofoed Nielsen, Christian Hansen","doi":"10.21769/BioProtoc.5776","DOIUrl":"10.21769/BioProtoc.5776","url":null,"abstract":"Progressive neurodegeneration linked to the accumulation of misfolded proteins is a hallmark of several neurodegenerative disorders, including Parkinson's disease, Huntington's disease, and Alzheimer's disease. Dysfunction in the protein homeostasis machinery correlates with pathology. The chaperone protein DNAJB6 is expressed in neurons and oligodendrocytes and has been shown to play a key role in preventing amyloid aggregation by binding to amyloidogenic proteins and facilitating their refolding or degradation, in cooperation with other chaperones. Here, we describe a simple and feasible assay that enables high-throughput screening for DNAJB6 activity in a plate reader format. We use genetically engineered HEK293 cells that stably express DNAJB6 fused to either CFP or YFP. These cells can be plated into multi-well plates, and the fluorescence resonance energy transfer (FRET) signal can be measured for analysis of DNAJB6 dimerization, which is linked to DNAJB6 activity. The protocol can be used for drug screening and to identify compounds that increase DNAJB6 dimerization, and can serve as a starting point for finding new medicines that act through modulating DNAJB6 activity. Key features • The protocol requires a plate reader capable of FRET analysis and bandwidth adjustment for CFP/YFP separation. It was developed using a CLARIOstar plate reader. • The protocol requires access to the authors' FRET DNAJB6 cell line or equivalent cells with stable expression of CFP/YFP-DNAJB6. • The assay measures DNAJB6 dimerization and can potentially be adapted to other proteins whose functional state is linked to dimerization activity. • The protocol is useful for compound screening purposes and requires pre-existing knowledge of basic cell culture.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5776"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454940/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708948","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 for In Vitro Activation of Jurkat E6-1 Cells Using Recombinant Human Galectin. 重组人凝集素体外激活Jurkat E6-1细胞的方法

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5772

Ruei-De Yan, Ling Mao, Yi-Jing Chen, Chien-Hui Lo, Yi-Chang Liu

{"title":"Protocol for In Vitro Activation of Jurkat E6-1 Cells Using Recombinant Human Galectin.","authors":"Ruei-De Yan, Ling Mao, Yi-Jing Chen, Chien-Hui Lo, Yi-Chang Liu","doi":"10.21769/BioProtoc.5772","DOIUrl":"10.21769/BioProtoc.5772","url":null,"abstract":"Surface receptor engagement governs T-cell activation. Since these surface receptors undergo extensive glycosylation, lectin-mediated crosslinking of these glycosylated surface receptors has the potential to modulate signaling. Here, we systematically evaluate the abilities of recombinant human galectins in triggering immune responses. We describe how to apply the human galectins to modulate Jurkat E6-1 cell activation by measuring the expression level of cellular surface CD69 and the mRNA of IL-2. To validate the protocol, we confirmed that galectin-3 and galectin-8 variants 1 and 2 reproducibly induce CD69 and IL-2 expression on Jurkat E6-1 cells. Our approach offers a galectin-based toolset to study how glycosylation modulates human adaptive immunity. Key features • Systematic screening of the recombinant human galectin 1, 3, 7, 8 (both of variant 1 and variant 2) for Jurkat E6-1 cell modulatory activity. • Mechanism-based approach utilizing glycan crosslinking to induce receptor engagement. • Standardized workflow to evaluate extracellular galectin-driven immune activation.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5772"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454943/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708914","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

Liposome-based Expression of the PIEZO1 Sensor GenEPi in Hippocampal Neurons in Organotypic Slices. 基于脂质体的PIEZO1传感器基因在海马神经元器官型切片中的表达。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5780

Anya Bhavnani, Olga Kopach

{"title":"Liposome-based Expression of the PIEZO1 Sensor GenEPi in Hippocampal Neurons in Organotypic Slices.","authors":"Anya Bhavnani, Olga Kopach","doi":"10.21769/BioProtoc.5780","DOIUrl":"10.21769/BioProtoc.5780","url":null,"abstract":"Expressing large DNA constructs in the native three-dimensional brain microenvironment remains technically challenging. Although viral vectors provide high transduction efficiency and cell-type selectivity, their genetic payload capacity is limited. Various non-viral approaches have been used in brain tissue, but they may compromise tissue viability or require specialised equipment, such as biolistic delivery or electroporation. We present an adapted protocol for delivering the large DNA vector encoding the optical PIEZO1 sensor GenEPi into brain tissue to enable sensor expression in pyramidal neurons. By applying DNA-Lipofectamine liposomes directly to the slice surface, we achieved efficient, minimally invasive transfection of pyramidal neurons in the CA1 and CA3 regions of organotypic hippocampal slices. PIEZO1 sensor expression was detectable as early as 7 days after transfection, increased with longer tissue maintenance, and was sustained for 3-4 weeks in vitro. This protocol describes a cost-effective, non-invasive approach that preserves cell viability and enables investigation of PIEZO1-mediated mechanotransduction in a native brain microenvironment. Key features • DNA-Lipofectamine liposomes are applied directly to slice surface, enabling efficient transfection of superficial hippocampal neurons, important for imaging experiments performed using upright microscope systems. • The protocol provides a cost-effective gene delivery approach that requires only small volumes of DNA and transfection reagent. • Robust expression of the PIEZO1 sensor GenEPi is achieved within a relatively short time (approximately 1 week after transfection). • The method is compatible with long-term tissue maintenance, with neuronal viability and GenEPi expression maintained for up to 3-4 weeks after transfection.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5780"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454942/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708930","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

direct Stochastic Optical Reconstruction Microscopy to Determine the Oligomeric State of Proteins on the Plasma Membrane and Their Accessibility for Immunotherapeutic Antibodies. 直接随机光学重建显微镜测定蛋白在质膜上的寡聚状态及其对免疫治疗抗体的可及性。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5767

Patrick Eiring, Sören Doose, Nele Bauer, Markus Sauer

{"title":"direct Stochastic Optical Reconstruction Microscopy to Determine the Oligomeric State of Proteins on the Plasma Membrane and Their Accessibility for Immunotherapeutic Antibodies.","authors":"Patrick Eiring, Sören Doose, Nele Bauer, Markus Sauer","doi":"10.21769/BioProtoc.5767","DOIUrl":"10.21769/BioProtoc.5767","url":null,"abstract":"Super-resolution fluorescence microscopy enables the visualization of protein structures at nanometer resolution, providing insights into receptor organization on the plasma membrane that are essential for the development and optimization of immunotherapies. In this context, monoclonal antibodies are employed, which typically bind only a subset of available membrane receptors, due to steric hindrance or otherwise limited epitope accessibility, to quantify the accessible targets. These accessible targets, rather than the total receptor density, are critical for determining therapeutic efficacy. Here, we present a simplified, robust protocol to quantify antibody-accessible endogenous receptors using monoclonal antibodies directly labeled with fluorescent dyes in combination with total internal reflection fluorescence (TIRF) direct stochastic optical reconstruction microscopy (dSTORM). The method employs optimized labeling and fixation conditions to preserve the native receptor distribution, enabling precise quantification of accessible receptors and their stoichiometry at single-molecule resolution. Omitting secondary antibodies and minimizing fixation-induced artifacts prevents artificial clustering and maintains the physiological binding pattern of therapeutic antibodies. The standardized workflow delivers therapy-relevant information about receptor accessibility and organization underlying therapeutic antibody binding, thereby advancing the mechanistic understanding of immunotherapy resistance and personalized treatment strategies across diverse membrane protein targets. Key features • Influence of fixation conditions on receptor epitope accessibility. • Use of therapeutic antibodies for quantitative estimation of receptor availability relevant to immunotherapy. • Fluorophore localizations provide information on antibody binding events below the optical resolution limit.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5767"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13461754/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148726620","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

Sample Preparation for Imaging-Based Spatial Transcriptomics in Rigid Plant Tissues (Roots, Shoots). 基于成像的植物刚性组织（根，芽）空间转录组学样品制备。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5779

Hanhong Liu, Jingyuan Zhang, Mingyuan Zhu

{"title":"Sample Preparation for Imaging-Based Spatial Transcriptomics in Rigid Plant Tissues (Roots, Shoots).","authors":"Hanhong Liu, Jingyuan Zhang, Mingyuan Zhu","doi":"10.21769/BioProtoc.5779","DOIUrl":"10.21769/BioProtoc.5779","url":null,"abstract":"Plant roots dynamically respond to environmental changes and serve as an ideal system for studying cell development and gene regulation. Recent advances in imaging-based spatial transcriptomics have enabled high-resolution mapping of gene expression while preserving spatial context. However, existing sample preparation techniques remain inadequate for handling rigid plant tissues such as crop roots. Here, we present a detailed and practical protocol for preparing rigid plant tissue samples for imaging-based spatial transcriptomics. The workflow ensures effective tissue handling while maintaining RNA integrity and spatial organization. Within approximately eight days, samples can be processed and mounted onto commercial slides, making them ready for subsequent probe hybridization and imaging. This protocol also includes an integrated sample attachment test performed to assess slide quality. It has been optimized to produce consistent and reliable results across experiments. Overall, our method provides a robust solution for spatial transcriptomic analysis in rigid plant tissues, facilitating broader application of these technologies in plant research. Key features • Builds upon the method developed by Zhu et al. [1] and introduces an optimized sample preparation protocol for imaging-based spatial transcriptomics in rigid rice roots. • Ensures effective tissue fixation and sectioning, while preserving RNA integrity and spatial organization. • Includes an integrated sample attachment test to assess the adhesion of tissue sections to commercial slides. • Requires approximately 8 days to complete the sample preparation, with another 6 days for the attachment test.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5779"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454941/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708958","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

Visualizing the Osteocyte Lacuno-Canalicular System via a Rapid 10-Minute Silver Nitrate Staining Method. 通过快速10分钟硝酸银染色法观察骨细胞腔隙-小管系统。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5784

Jinlian Wu, Weiwei Dai, Libo Wang

{"title":"Visualizing the Osteocyte Lacuno-Canalicular System via a Rapid 10-Minute Silver Nitrate Staining Method.","authors":"Jinlian Wu, Weiwei Dai, Libo Wang","doi":"10.21769/BioProtoc.5784","DOIUrl":"10.21769/BioProtoc.5784","url":null,"abstract":"The conventional Ploton silver method employs a high-concentration 50% (w/v; 2.943 mol/L) silver nitrate solution for histological staining and characterization of the osteocyte lacuno-canalicular system (LCS). However, it is limited by prolonged staining times (55 min) and by risks of LCS ultrastructural damage and/or incomplete impregnation. To address these limitations, we developed the Wu-Wang silver nitrate staining method, which uses a 1 mol/L silver nitrate solution under elevated temperature (50-70 °C) to achieve rapid, effective, and high-contrast visualization of the osteocyte LCS within 10 min. We further demonstrate that this novel method enables robust LCS visualization across multiple vertebrate species. Compared with the Ploton method, the Wu-Wang method substantially reduces staining time and overcomes staining limitations inherent to prolonged exposure to concentrated silver nitrate solutions. This rapid and efficient staining method supports more accurate quantitative analysis of LCS morphology and facilitates systematic investigation of osteocyte and LCS morphogenesis, as well as the pathological mechanisms underlying bone and joint disease. Key features • A novel rapid method uses 1 mol/L silver nitrate at 50-70 °C, enabling high-contrast LCS staining within 10 min. • A rapid silver nitrate staining method provides superior, consistent LCS visualization across multiple vertebrate species. • The novel method serves as a fast, efficient histological tool to investigate osteocytes, LCS, and bone-joint pathology.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5784"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454885/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708925","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

Dual Color tau-STED Super Resolution Microscopy in Arabidopsis Root Tip. 拟南芥根尖双色陶氏超分辨显微镜研究。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5774

Louise Fougère, Christel Poujol, Yohann Boutté, Magali Grison

{"title":"Dual Color tau-STED Super Resolution Microscopy in Arabidopsis Root Tip.","authors":"Louise Fougère, Christel Poujol, Yohann Boutté, Magali Grison","doi":"10.21769/BioProtoc.5774","DOIUrl":"10.21769/BioProtoc.5774","url":null,"abstract":"Super-resolution microscopy has transformed our ability to visualize subcellular structures, but its application in plant biology remains challenging due to the optical complexity of plant tissues. Here, we present a detailed protocol for tau-STED microscopy (Leica Microsystems), which combines stimulated emission depletion (STED) with fluorescence lifetime imaging (FLIM) to achieve nanoscale resolution while minimizing phototoxicity. This method leverages time-correlated single-photon counting (TCSPC) to separate fluorescence signals based on their lifetimes, enhancing signal specificity and enabling the visualization of elusive subcellular compartments in Arabidopsis thaliana root tips. The protocol covers sample preparation, fluorophore selection, microscope configuration, image acquisition, and data analysis, providing a step-by-step guide to optimize tau-STED imaging for plant cell biology. By addressing the unique challenges of plant tissue imaging, such as autofluorescence, refractive index mismatches, and light scattering, this approach facilitates super-resolution imaging of intracellular structures, including the plant endoplasmic reticulum-Golgi intermediate compartment (ERGIC). This protocol is designed to be accessible to researchers with basic microscopy experience and offers a robust framework for exploring subcellular dynamics in plants with unprecedented detail. Key features • tau-STED integrates STED signals with fluorescence lifetime via phasor analysis at confocal speeds, enabling low-noise super-resolution imaging. • Morphometry analysis workflow at super resolution.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5774"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454947/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708919","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

In Vivo and In Vitro SUMOylation Assays in Arabidopsis. 拟南芥体内和体外sumo酰化分析。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5769

Xiao Liu, Shan Tang, Xupeng Guo, Chengming Fan, Zanmin Hu

{"title":"In Vivo and In Vitro SUMOylation Assays in Arabidopsis.","authors":"Xiao Liu, Shan Tang, Xupeng Guo, Chengming Fan, Zanmin Hu","doi":"10.21769/BioProtoc.5769","DOIUrl":"10.21769/BioProtoc.5769","url":null,"abstract":"Small ubiquitin-like modification (SUMOylation) is a crucial post-translational modification that modulates protein stability, localization, and interaction dynamics. Despite the identification of thousands of putative small ubiquitin-like modifier (SUMO) substrates, functional validation remains challenging due to the low abundance and highly dynamic nature of SUMOylated proteins. Here, we present a protocol for detecting protein SUMOylation, integrating bioinformatic site prediction, and rapid substrate screening via in vivo tobacco transient expression and in vitro E. coli assay, followed by precise validation using transgenic Arabidopsis lines. However, detection of low-abundance SUMOylated proteins may require coupling with mass spectrometry, and the in vitro system does not fully recapitulate the complex regulatory network in vivo. This workflow provides a useful tool for studying SUMOylation in plants. Key features • Integrates bioinformatic prediction, in vitro validation, and in vivo confirmation for SUMOylation analysis. • E. coli co-expression system enables rapid SUMOylation detection without protein purification. • Arabidopsis transgenic line system confirms SUMOylation under physiological conditions. • The protocol is applicable to most Arabidopsis proteins.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5769"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13461753/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148726959","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

Generation of 3D Hemogenic Gastruloids From Mouse Embryonic Stem Cells. 从小鼠胚胎干细胞中生成三维造血原胃样细胞。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5777

Ylenia Cicirò, Cristina Pina, Denise Ragusa

{"title":"Generation of 3D Hemogenic Gastruloids From Mouse Embryonic Stem Cells.","authors":"Ylenia Cicirò, Cristina Pina, Denise Ragusa","doi":"10.21769/BioProtoc.5777","DOIUrl":"10.21769/BioProtoc.5777","url":null,"abstract":"Embryonic blood formation encompasses the independent generation of different cell types in distinct cellular and anatomical environments, reflecting highly coordinated specific hierarchies of interacting tissues. Despite widespread use of embryonic stem cells (ESC) and induced pluripotent stem cell (iPSC)-based models to attempt to capture blood development in vitro and generate hematopoietic stem cells (HSC), a system that fully captures the spatial and temporal complexity of embryonic hematopoiesis is still lacking. In recent years, gastruloid models have emerged as powerful representations of early development, demonstrating self-organizing behaviors such as symmetry breaking, elongation, multi-axis formation, somitogenesis, and early organogenesis, with striking parallels to embryonic processes. Here, we present a protocol to generate hemogenic gastruloids (haemGx) from mouse ESC (mESC) that closely recapitulates the multi-stage, multi-niche process of blood formation and generates developmentally accurate hematopoietic progenitors. The haemGx model has been proven valuable in understanding embryonic hematopoiesis, as well as an in vitro model of forms of infant leukemia with an embryonic, in utero origin. Key features • The haemGx protocol allows the generation of developmentally accurate endothelial and hematopoietic precursor and progenitor cell types. • The haemGx protocol achieves a level of spatiotemporal control that closely recapitulates key aspects of embryonic development. • The haemGx protocol is compatible with multiple mESC lines, enabling reproducible generation of developmental blood cell types across different genetic backgrounds. • At endpoint, individual haemGx reaches ~800-1,000 μm in diameter and contains 30,000/50,000 cells. The 96-well format allows for upscaling and high-throughput applications.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5777"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13454945/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708882","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