Bio-protocol最新文献_第7页

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

DepStep: An Efficient One-Step rRNA Depletion Workflow for RNA Sequencing in Non-model Organisms. DepStep：用于非模式生物RNA测序的高效一步rRNA耗尽工作流。

IF 1.3

Bio-protocol Pub Date : 2026-08-05 DOI: 10.21769/BioProtoc.5764

Suleman M Qasim, Peter L Sarin

{"title":"DepStep: An Efficient One-Step rRNA Depletion Workflow for RNA Sequencing in Non-model Organisms.","authors":"Suleman M Qasim, Peter L Sarin","doi":"10.21769/BioProtoc.5764","DOIUrl":"10.21769/BioProtoc.5764","url":null,"abstract":"RNA sequencing (RNA-seq) has revolutionized transcriptomics, ribosome footprinting, and polysome profiling, providing a wealth of data. Many RNA-based omics typically remove ribosomal RNA (rRNA) or select for messenger RNA (mRNA) prior to sequencing, thereby enriching reads that map to the translationally active part of the transcriptome. Prokaryotic mRNA lacks the 3' polyadenylated tail, which excludes the use of poly(A)-based selection methods. While commercial rRNA depletion products exist for prokaryotes, their proprietary nature and potential inefficiency with non-model organisms are factors that may limit broad-scale application. To mitigate this issue, we designed DepStep, a consolidated workflow for one-step rRNA depletion using species-specific biotinylated antisense probes for selective hybridization and removal of the target rRNA molecules. As a proof-of-concept, RNA-seq libraries of the psychrophilic gram-negative bacterium Shewanella glacialimarina TZS-4T were prepared using both DepStep and a commercial rRNA depletion kit for gram-negative bacteria, to which DepStep was benchmarked. DepStep compares favorably to the commercial depletion kit; it removes >98.6% of the rRNA content in the sample, resulting in sequencing libraries where the coding DNA sequence (CDS) reads account for >80% of the total read count. Importantly, DepStep's cost-per-sample is three times lower than the commercial kit, establishing DepStep as a simple yet cost-effective alternative to commercial solutions. Key features • DepStep is a comprehensive workflow for one-step rRNA depletion in non-model species. • Guidelines for design and implementation of specific biotinylated antisense probes. • Compares favorably in benchmarking to commercial products. • Alternative for budget-conscious labs with 3× lower cost-per-sample.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 15","pages":"e5764"},"PeriodicalIF":1.3,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13461757/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148724647","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 Streamlined and Time-Saving Approach to Generate HLA-DR15 MHC Class II Tetramers via In Vivo Biotinylation. 通过体内生物素化生成HLA-DR15 MHC II类四聚体的简化和节省时间的方法。

IF 1.3

Bio-protocol Pub Date : 2026-07-20 DOI: 10.21769/BioProtoc.5755

Xue-Yao Zhao, Heng-Hui Li, Hong-Yan Ma, Bin Yang, Ru-Yi Qian, Xiang Zhang, Ling-Jie Luo, Yan-Wei Wu, Liang Chen

{"title":"A Streamlined and Time-Saving Approach to Generate HLA-DR15 MHC Class II Tetramers via In Vivo Biotinylation.","authors":"Xue-Yao Zhao, Heng-Hui Li, Hong-Yan Ma, Bin Yang, Ru-Yi Qian, Xiang Zhang, Ling-Jie Luo, Yan-Wei Wu, Liang Chen","doi":"10.21769/BioProtoc.5755","DOIUrl":"10.21769/BioProtoc.5755","url":null,"abstract":"This protocol describes an optimized strategy for the efficient generation of peptide-loaded major histocompatibility complex (MHC) class II (pMHC) tetramers, which are essential tools for detecting and characterizing antigen-specific T cells in immunological research. Traditional methods require separate expression of MHC proteins followed by in vitro biotinylation-a multi-step process that is time-consuming and prone to protein loss. Here, we present an integrated approach based on co-expression of MHC monomers and BirA biotin ligase in Expi293F T cells, enabling site-specific biotinylation in vivo during protein synthesis. At the same time, the incorporation of a thrombin-cleavable class II-associated invariant chain peptide (CLIP) peptide into the MHC construct allows flexible loading of any antigenic peptide of interest without the need for re-cloning or re-expression of the MHC molecule. Pre-biotinylated MHC molecules are subsequently purified, loaded with antigenic peptides, and assembled into fluorescent tetramers via streptavidin conjugation. This streamlined workflow significantly reduces handling steps, improves protein yield, and enhances reproducibility. The resulting tetramers are suitable for sensitive detection and isolation of antigen-specific T cells by flow cytometry, supporting applications in T-cell immunogenicity studies, vaccine development, and autoimmune disease research. Key features • One-step in vivo biotinylation. • Eliminates purchased BirA enzyme, saves ~16 h, and reduces protein loss. • CLIP-based peptide exchange without re-cloning. • Produces antigen-specific tetramers validated by flow cytometry.","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 14","pages":"e5755"},"PeriodicalIF":1.3,"publicationDate":"2026-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13403119/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148611162","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