Bio-protocol最新文献

{"title":"Humanizing Antibodies and Nanobodies From Scratch With HuDiff.","authors":"Yang Nan, Hongshuai Sun, Bo Zhang, Yue Yang, Jianxin He, Qifeng Bai","doi":"10.21769/BioProtoc.5816","DOIUrl":"10.21769/BioProtoc.5816","url":null,"abstract":"<p><p>Antibody (Ab) and nanobody (Nb) humanization is essential for reducing immunogenicity in therapeutic applications. HuDiff is an adaptive autoregressive diffusion approach that generates humanized antibodies and nanobodies from scratch using only complementarity-determining region sequences as input, eliminating the need for preexisting human templates. The method follows a two-stage training pipeline: pretraining on human antibody sequences to learn framework region patterns, followed by fine-tuning on target-species sequences. HuDiff-Ab processes paired heavy and light chains for conventional antibodies, while HuDiff-Nb can incorporate a specialized inpainting mode to preserve critical nanobody framework residues. This protocol provides a complete step-by-step guide for implementing HuDiff, covering data preparation, model training, and sequence generation. Key features • Requires only CDR sequences as input and does not require human template selection. • Uses a two-stage training strategy, with pretraining on human antibody sequences and fine-tuning on target-species sequences guided by humanness scores. • HuDiff-Ab humanizes paired heavy and light chains simultaneously, whereas HuDiff-Nb provides an inpainting mode to preserve key framework residues. • Generates multiple diverse humanized candidates for downstream experimental screening.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5816"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561673/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148932146","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}

{"title":"Generation of Budoids: 3D Multilineage Limb Models From Mouse Embryonic Stem Cells.","authors":"Kelly Hu, Can Aztekin","doi":"10.21769/BioProtoc.5808","DOIUrl":"10.21769/BioProtoc.5808","url":null,"abstract":"<p><p>Limb development requires the coordination of multiple cell types, including the limb bud mesoderm and surface ectoderm, by the apical ectodermal ridge (AER), a specialized signaling center secreting numerous morphogens. Characterizing these cell-cell interactions is crucial for understanding limb morphogenesis, but they are challenging to study in vivo. Furthermore, existing in vitro models do not capture the multilineage complexity of the limb. We recently developed a robust 7-day differentiation protocol using mouse embryonic stem cells (mESCs) to generate heterogeneous cultures containing cells with characteristics of the limb bud mesoderm, surface ectoderm, and AER. Dissociating and reaggregating these cultures in low attachment 96-well plates forms budoids, organoids that display certain limb bud-like features. Budoids undergo chondrogenesis-mediated symmetry breaking and elongation within 5 days of culture. Altogether, our protocols have enabled the study of cell-cell interactions in limb development and provide an easily scalable model adaptable for various applications, including drug testing and congenital disorder modeling. Key features • Induction of heterogeneous 2D cultures containing three main cell types of the developing limb bud from mESCs. • Free-floating budoid cultures achieve 3D morphogenesis without manual manipulation or embedding. • Highly scalable protocol, per differentiation experiment yielding > 9 × 10⁶ cells, sufficient for generating >1,000 budoids.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5808"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561693/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148957572","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}

{"title":"scDynaBar: A Step-By-Step Experimental and Computational Guide for Time-Resolved CRISPR Barcoding at Single-Cell Resolution.","authors":"Yolanda Andres-Lopez, Carla El Khouri-Gonzalez, Irene Hernando-Herraez","doi":"10.21769/BioProtoc.5793","DOIUrl":"10.21769/BioProtoc.5793","url":null,"abstract":"<p><p>CRISPR-Cas9 barcoding technologies enable cells to record molecular events as permanent genetic changes that can be read out retrospectively. This protocol describes the implementation of a CRISPR-based recording system that gradually accumulates mutations over extended periods and is compatible with standard single-cell RNA sequencing (scRNA-seq) workflows. By temporally regulating CRISPR activity, the system generates mutational barcodes that can be captured together with individual cell transcriptomes. These barcodes are subsequently decoded using computational reconstruction approaches to infer temporal information, enabling the joint analysis of cellular states and time-resolved molecular histories. This approach provides a single-cell-compatible framework for studying dynamic biological processes in heterogeneous mouse embryonic stem cell (mESC)-derived systems, with potential extension to other biological systems. Key features • Extended temporal recording: Self-targeting guide RNAs drive progressive and cumulative barcode divergence over time. • Simultaneous barcode and transcriptome detection: Joint recovery of genetic barcodes and whole transcriptomes from the same single cell using standard scRNA-seq workflows. • In inducible scDynaBar designs: Cas9 barcode editing can be coupled to specific biological stimuli or cell-state transitions, e.g., transition of mESCs into the 2C-like state.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5793"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13558868/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148932170","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}

{"title":"From Bacterial Cellulose Production by <i>Komagataeibacter xylinus</i> to Bacterial Cellulose Nanoparticles: A Standardized Enzymatic Approach.","authors":"Martina Schibeci, Rosa Gaglione, Erika Piccolo, Bartolomeo Della Ventura, Angela Arciello","doi":"10.21769/BioProtoc.5812","DOIUrl":"10.21769/BioProtoc.5812","url":null,"abstract":"<p><p>Bacterial cellulose (BC) is a renewable biopolymer valued for its exceptional purity, biocompatibility, and mechanical strength, with broad applications in biomedicine and sustainable materials. However, achieving reproducible BC production and downstream processing remains a major challenge. Inoculum preparation is particularly difficult to standardize because cellulose-producing strains form pellicles that sequester cells, making optical density measurements unreliable. In addition, recovery and drying procedures can alter fiber accessibility, and enzymatic hydrolysis conditions are often inconsistently defined and lack proper enzyme activity assessment. These issues contribute to substantial variability in BC-derived nanoparticle yields. This protocol describes the production of BC from <i>Komagataeibacter xylinus</i> DSMZ 6513, including culture medium preparation, inoculum generation, and scaling up under static cultivation conditions. It further details BC pellicle purification using NaOH, followed by pulping, freeze-drying, and milling to ensure material stability during storage and use. BC hydrolysis is performed with commercially available cellulase from <i>Trichoderma reesei</i>, with enzyme activity quantified prior to each reaction to ensure reproducibility. This standardized approach enables the reproducible production of bacterial cellulose nanoparticles (BCNPs). The protocol also includes minimal morphological characterization methods. By standardizing culture, recovery, and hydrolysis steps, the workflow reduces experimental variability and improves comparability across laboratories. Overall, it provides an accessible and reproducible method for generating BC and BCNPs of consistent quality without the need for specialized instrumentation. Key features • Standardized inoculum preparation that does not rely on optical density measurements, ensuring reproducible starting conditions for cellulose-producing strains. • Controlled recovery and freeze-drying procedures that preserve fiber accessibility and promote consistent enzymatic hydrolysis outcomes. • Use of commercially available <i>Trichoderma reesei</i> cellulase (≥700 units/g), with enzyme dosage defined by measured activity according to a publicly available Megazyme protocol. • Minimal but robust morphological characterization methods to assess material quality.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5812"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561677/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148946387","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}

{"title":"Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron-Myotube Co-cultures.","authors":"Anand Ganapathy Subramaniam, Lucas Keniger de Andrade Gensas, Tal Gradus-Pery, Eran Perlson","doi":"10.21769/BioProtoc.5801","DOIUrl":"10.21769/BioProtoc.5801","url":null,"abstract":"<p><p>Amyotrophic lateral sclerosis (ALS) is characterized by early and spatially restricted pathology in motor axons, including distal degeneration and accumulation of aggregation-prone proteins such as TDP-43. However, a major limitation in the field has been the lack of approaches that enable robust, quantitative, and compartment-specific analysis of these early axonal events, particularly in human-relevant systems. Here, we describe an integrated experimental and analytical framework that enables quantitative dissection of axonal degeneration and protein aggregation, specifically within distal motor axons. By combining compartmentalized human co-cultures with a dedicated image analysis strategy, this approach enables selective and quantitative analysis of pathological processes specifically within axons, independent of surrounding tissues such as muscle and other cellular compartments. This framework captures both structural degeneration and protein aggregation dynamics at subcellular resolution, enabling spatially resolved quantitative analysis of disease-relevant changes along axons. Importantly, the analytical framework is not limited to TDP-43 but is broadly applicable to diverse aggregation-prone proteins, thereby providing a generalizable platform to study axonal pathology across neurodegenerative diseases. Together, this work provides a scalable approach for investigating axonal pathology as an early and measurable feature of neurodegeneration, with potential applications in mechanistic studies and therapeutic targeting in ALS and related disorders. Key features • Compartmentalized human induced pluripotent stem cell (iPSC)-derived motor neuron-myotube co-cultures for modeling distal axonal pathology. • Microfluidic separation of somatic and distal axonal compartments enabling spatial perturbation and analysis. • Quantitative imaging of neurofilament heavy chain (NFH)-associated axonal degeneration and pTDP-43 accumulation. • Semi-automated workflow for a reproducible, scalable, and modular pipeline for image quantification.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5801"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561694/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148957549","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}

{"title":"A Modified Slide-Embedded Scanning Electron Microscopy Preparation Method to Visualize Antagonistic Interactions Between <i>Trichoderma viride</i> and <i>Fusarium</i> sp.","authors":"Sandra Tomichen, Shweta Panchal","doi":"10.21769/BioProtoc.5805","DOIUrl":"10.21769/BioProtoc.5805","url":null,"abstract":"<p><p>Mycoparasitism is an important mechanism of fungal antagonism in which one fungus parasitizes another. This type of interaction plays a major role in the biocontrol activity of <i>Trichoderma</i> spp. against phytopathogenic fungi. Detailed visualization of these interactions is essential for understanding the structural mechanisms involved in fungal antagonism, including hyphal attachment, coiling, penetration, and cellular distortion. Scanning electron microscopy (SEM) is widely used for structural examination of fungal interactions; however, conventional preparation methods such as filter paper systems, membrane overlays, and agar block techniques often result in structural distortion, fragile sample handling, and difficulty in locating defined interaction zones. Here, we describe a modified slide-embedded technique for SEM visualization of mycoparasitic interactions between filamentous fungi. The protocol is adapted from previously reported slide culture approaches and involves embedding pre-cut sterile glass slide fragments directly into potato dextrose agar (PDA), followed by sequential inoculation of <i>Fusarium</i> sp. and <i>Trichoderma viride</i>. Fungal interactions occurring directly on the glass surface are subsequently subjected to fixation with 2.5% glutaraldehyde, graded ethanol dehydration, sputter coating, and SEM observation. Compared with conventional methods, the present approach provides improved handling stability, better preservation of native hyphal architecture, reduced deformation during processing, and easier localization of interaction zones during microscopy. The protocol also enables clear visualization of early antagonistic events such as hyphal coiling, penetration, and surface colonization. Due to its simplicity, reproducibility, and minimal technical complexity, this method serves as a practical and efficient approach for SEM-based investigation of fungal-fungal interactions and can be readily adapted for studying diverse mycoparasitic systems. Key features • A simple, cost-effective method to visualize filamentous fungus-fungus interactions, particularly mycoparasitism, using minimal reagents and basic laboratory equipment. • A standardized inoculation strategy with pathogen inoculation 48 h before the mycoparasite ensures reproducible fungal interaction and mycoparasitic development. • Blunt-cut square glass slide pieces embedded in PDA enable easy sample handling, storage, transport, and direct preparation for scanning electron microscopy. • SEM-compatible fixation and ethanol dehydration preserve fungal ultrastructure, enabling high-resolution visualization of hyphal coiling, distortion, and other mycoparasitic interactions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5805"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561691/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148957574","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}

{"title":"Identifying D-Group Mitogen-Activated Protein Kinases as Substrates of <i>Arabidopsis</i> Tyrosine Phosphatase RLPH2 Using Phospho-Tyrosine Peptide Enrichment.","authors":"Anne-Marie Labandera, Brooklyn Kurucz, R Glen Uhrig, Greg B Moorhead","doi":"10.21769/BioProtoc.5810","DOIUrl":"10.21769/BioProtoc.5810","url":null,"abstract":"<p><p>Identifying substrates of protein phosphatases has been technically challenging and has hampered progress in the field of plant sciences. Small molecule inhibitors of protein phosphatases have aided in uncovering classes of phosphatases that target substrates, but that too has severe limitations. Here, we describe a method that enriches phosphorylated substrates using TiO₂ and phospho-tyrosine antibodies in phosphatase knockout lines of <i>Arabidopsis thaliana</i>. When compared to wild-type plants, this approach permits identification of putative substrates and specific phosphorylation sites by mass spectrometry, allowing for further in vitro or functional validation. The key to the approach described here is the use of phosphatase knockout lines to maintain substrates in a phosphorylated state and using phospho-tyrosine antibodies to enrich for tyrosine phosphorylated peptides. Key features • Requires genetic knockout lines for the protein phosphatase of interest to uncover increased phosphorylation of putative substrates compared to wild type. • Phospho-peptides are (quantitatively) compared using mass spectrometry. • Tyrosine phosphorylated peptides are immunoprecipitated after TiO₂ phospho-peptide enrichment. • Putative substrates identified with this protocol can later be validated with in vitro assays using synthetic phospho-peptides and/or phospho-proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5810"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561676/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148946313","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}

{"title":"An Automated, Ventana Discovery Platform-based Imaging Workflow for Simultaneous Quantification of B Cells, Plasma Cells, and Plasmablasts in FFPE Human Tissues.","authors":"Patrick S Chang, Caleb Chan, Rajiv Jesudason, Sandra Rost, Cary D Austin","doi":"10.21769/BioProtoc.5797","DOIUrl":"10.21769/BioProtoc.5797","url":null,"abstract":"<p><p>Accurate, sensitive quantification of B-lineage cells is critical for pharmacodynamic evaluation of B cell-targeted therapies in lupus nephritis (LN) clinical trials. While high-dimensional discovery platforms offer broad profiling, they often lack the sensitivity, quantitative rigor, and throughput needed for precise cell enumeration in renal trial needle biopsies. Traditional immunostaining is hampered by CD20-directed therapeutic masking or downregulation, inadequate sensitivity of CD19 in FFPE tissue, and confounding renal tubular CD138 expression. This protocol details an automated, fit-for-purpose, 5-plex sequential tyramide signal amplification (TSA)-based immunofluorescence assay (CD38, CD79a, CD19, Ki-67, CD138) developed on the Ventana Discovery Ultra platform for deployment on single tissue sections. The workflow anchors B-cell detection on CD79a to ensure sensitivity and utilizes CD38 as an obligate co-marker for CD138+ antibody-secreting cells (ASCs) to definitively exclude the CD138+ epithelial background. Following acquisition via fluorescence whole-slide imaging, a digital analysis pipeline utilizing InstanSeg-based automated segmentation rigorously classifies cell phenotypes to generate precise spatial densities (cells/mm²). This validated protocol maximizes data yield from scarce clinical biopsies while providing high-precision quantitative monitoring of longitudinal therapeutic depletion in the renal microenvironment. Key features • Automated trial scalability: Details an automated, TSA-based 5-plex assay optimized for the Ventana Discovery Ultra platform, ensuring high-throughput, reproducible B-lineage quantification in multicenter cohorts. • Robust lineage identification: Establishes CD79a as an anchor for sensitive B-cell detection in FFPE tissue, avoiding CD20 therapeutic masking/downregulation and CD19 epitope instability. • High-specificity ASC enumeration: Resolves confounding CD138+ renal tubular background using an obligate CD38/CD138 co-labeling strategy to definitively identify ASCs within the renal microenvironment. • Single-section phenotypic resolution: Combines multiplexing and InstanSeg segmentation on one 4-μm section to simultaneously quantify B-lineage subsets in tissue-limited renal needle biopsies.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 17","pages":"e5797"},"PeriodicalIF":1.3,"publicationDate":"2026-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561952/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148957503","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}

{"title":"Stepwise Generation of Vascularized Multilayered 3D Organotypic Skin Models.","authors":"Haiwei Zhai, Xiaowei Jin, Meghan Biegert, Brandyn Zahs, Ruiguo Yang, Fanben Meng","doi":"10.21769/BioProtoc.5802","DOIUrl":"10.21769/BioProtoc.5802","url":null,"abstract":"<p><p>Skin models play critical roles in understanding disease mechanisms and advancing therapeutic development. However, conventional systems based on 2D cell cultures, in vivo animal models, and ex vivo tissue explants are limited by insufficient physiological complexity, interspecies differences, and restricted accessibility, respectively. Advances in biofabrication technologies have enabled the engineering of 3D skin equivalents that better balance biological complexity and experimental scalability. Here, we present a biofabrication protocol inspired by the regenerative processes of wound healing to construct vascularized 3D organotypic skin models in a stepwise manner. The approach integrates bioprinting for precise spatial organization of cellular compartments with guided cell self-organization to achieve native-like tissue complexity and heterogeneity. Through a programmable culture strategy, tissue maturation proceeds sequentially through keratinocyte proliferation and collective migration, microchannel endothelialization, basal-to-suprabasal differentiation, and progressive extracellular matrix remodeling within a fibrin-based scaffold. The resulting tissue constructs comprise stratified epidermal layers positioned atop a vascularized, fibroblast-remodeled dermal matrix. Beyond reproducing key structural features of human skin, this protocol recapitulates cellular processes associated with tissue regeneration, providing a dynamic platform for investigating disease pathogenesis, progression, and therapeutic responses. Key features • This protocol provides detailed procedures for preparing cells and biomaterials used in bioink formulation. • This protocol outlines a stepwise biofabrication process for integrating keratinocytes, fibroblasts, and endothelial cells into a multicellular skin model. • This protocol employs PolyJet 3D printing for tissue culture chamber fabrication and extrusion-based 3D bioprinting for spatial placement of cell-laden compartments. • This protocol leverages a dynamic, wound healing-inspired cell self-organization process under a programmable culture strategy to promote tissue maturation and architectural development.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5802"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507974/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835194","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}

{"title":"A Simple and Reproducible ImageJ Workflow for Measuring Areas of Irregularly Shaped Necrotic Lesions on Plant Leaves.","authors":"Houssam E S Bensedira, Omar K Chehaba","doi":"10.21769/BioProtoc.5788","DOIUrl":"10.21769/BioProtoc.5788","url":null,"abstract":"<p><p>Accurately quantifying the areas of necrotic lesions on plant leaves is essential for evaluating plant-pathogen interactions and disease resistance. Although digital image analysis methods using ImageJ are widely employed, they often require case-specific optimization and may not be readily applicable across different experimental conditions. Furthermore, many studies have used ImageJ for lesion measurement without providing methodological details, which limits reproducibility. Here, we present a simple, step-by-step ImageJ workflow for measuring irregular necrotic lesions using a standard personal computer and mouse. The procedure relies on manual lesion selection using the freehand selection tool, followed by Gaussian smoothing, binarization, and automated particle analysis to extract lesion area measurements. By balancing manual isolation with computational thresholding, this protocol eliminates the need for extensive parameter tuning. This approach provides an accessible, reliable alternative to time-consuming color thresholding methods, thereby improving transparency and reproducibility in lesion quantification. The workflow's reproducibility has been confirmed through both intra-user and inter-user analyses. Key features • Relies on simple manual freehand selection combined with minimal image processing, requiring only a standard computer and mouse without specialized software or advanced training. • Enables accurate quantification of irregular necrotic lesions in conditions where automated thresholding methods require time-consuming optimization. • Provides a fully detailed, reproducible ImageJ workflow addressing common gaps in published methods, facilitating direct implementation. • Demonstrates high reproducibility, validated by intra-user and inter-user statistical analyses, ensuring reliable lesion quantification regardless of the operator.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5788"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507966/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835635","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}