Bio-protocol最新文献

{"title":"Quantitative Estimation of Auxin, Siderophore, and Hydrogen Cyanide Production in Halo and Drought-Tolerant Bacterial Isolates for Cucumber Growth.","authors":"Zeinab Fotoohiyan, Ali Salehi Sardoei","doi":"10.21769/BioProtoc.5471","DOIUrl":"10.21769/BioProtoc.5471","url":null,"abstract":"<p><p>Salt-tolerant bacteria can enhance plant growth through various mechanisms, including the production of auxin, siderophores, hydrogen cyanide, and the solubilization of insoluble phosphate. This study investigated the production of these growth-stimulating factors by salt- and drought-tolerant bacteria isolated from the arid and saline farmlands of Jiroft. Initially, we screened for bacterial strains that exhibited the highest levels of these factors. We then evaluated their effects on improving the growth indices of cucumber seedlings. Additionally, we optimized the protocols for isolating auxin, siderophores, hydrogen cyanide, and phosphate solubilization, which can also be applied to other host rhizobacteria to assess their growth-promoting compounds. Key features • The most resistant bacterial isolates to salinity and drought are identified by adding salt and polyethylene glycol to the culture medium in laboratory conditions. • This protocol can be used to evaluate the production levels of IAA, siderophore, hydrogen cyanide, and phosphate solubilization by salt- and drought-tolerant bacteria. • This protocol can also be used to evaluating the plant growth-promoting ability of salt- and drought-tolerant bacteria under greenhouse conditions. • This protocol can be applied to other host rhizobacteria to assess their growth-promoting compounds.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5471"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514138/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282147","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 Intestinal Epithelial Monolayers From Single-Cell Dissociated Organoids.","authors":"Neta Felsenthal, Danijela Matic Vignjevic","doi":"10.21769/BioProtoc.5474","DOIUrl":"10.21769/BioProtoc.5474","url":null,"abstract":"<p><p>Intestinal organoids are generated from intestinal epithelial stem cells, forming 3D mini-guts that are often used as an in vitro model to evaluate and manipulate the regenerative capacities of intestinal epithelial stem cells. Plating 3D organoids on different substrates transforms organoids into 2D monolayers, which self-organize to form crypt-like regions (which contain stem cells and transit amplifying cells) and villus-like regions (which contain differentiated cells). This \"open lumen\" organization facilitates multiple biochemical and biomechanical studies that are otherwise complex in 3D organoids, such as drug applications to the cell's apical side or precise control over substrate protein composition or substrate stiffness. Here, we describe a protocol to generate homogenous intestinal monolayers from single-cell intestinal organoid suspension, resulting in de novo crypt formation. Our protocol results in higher viability of intestinal cells, allowing successful monolayer formation. Key features • This protocol requires preexisting experience in culturing mouse intestinal organoids. • This protocol requires preexisting experience in generating polyacrylamide (PAA) gels for culturing 2D monolayers. • This protocol generates intestinal monolayers that can be subjected to additional analysis, e.g., drug treatment, immunofluorescent staining, single-molecule fluorescent in-situ hybridization (smFISH), or live imaging.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5474"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514133/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282165","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":"Advancing 2-DE Techniques: High-Efficiency Protein Extraction From Lupine Roots.","authors":"Sebastian Burchardt, Patrycja Wojtaczka, Agata Kućko, Maciej Ostrowski, Emilia Wilmowicz","doi":"10.21769/BioProtoc.5461","DOIUrl":"10.21769/BioProtoc.5461","url":null,"abstract":"<p><p>Protein isolation combined with two-dimensional electrophoresis (2-DE) is a powerful technique for analyzing complex protein mixtures, enabling the simultaneous separation of thousands of proteins. This method involves two distinct steps: isoelectric focusing (IEF), which separates proteins based on their isoelectric points (pI), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins by their relative molecular weights. However, the success of 2-DE is highly dependent on the quality of the starting material. Isolating proteins from plant mature roots is challenging due to interfering compounds and a thick, lignin-rich cell wall. Bacterial proteins and metabolites further complicate extraction in legumes, which form symbiotic relationships with bacteria. Endogenous proteases can degrade proteins, and microbial contaminants may co-purify with plant proteins. Therefore, comparing extraction methods is essential to minimize contaminants, maximize yield, and preserve protein integrity. In this study, we compare two protein isolation techniques for lupine roots and optimize a protein precipitation protocol to enhance the yield for downstream proteomic analyses. The effectiveness of each method was evaluated based on the quality and resolution of 2-DE gel images. The optimized protocol provides a reliable platform for comparative proteomics and functional studies of lupine root responses to stress, e.g., drought or salinity, and symbiotic interactions with bacteria. Key features • Protocol tailored for isolating proteins from lupine roots, including those involved in symbiotic relationships with bacteria. • Our method is suitable for analyzing complex protein mixtures through IEF and SDS-PAGE for high-resolution separation. • Optimized precipitation method increases protein yield for downstream mass spectrometry and comparative proteomic analyses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5461"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514137/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282159","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":"Assessing Temperature-Dependent DNA Cleavage by CRISPR-Cas9.","authors":"Alexa L Knight, Jinping Luo, George P Lisi","doi":"10.21769/BioProtoc.5463","DOIUrl":"10.21769/BioProtoc.5463","url":null,"abstract":"<p><p>The RNA-guided CRISPR-Cas9 endonuclease has been a transformative tool for laboratory biochemistry with huge potential as a precision therapeutic. This tool site-specifically cleaves double-stranded DNA following the recognition of a unique protospacer-adjacent motif (PAM). Activation of the protein-nucleic acid Cas complex has also been widely recognized to feature an allosteric mechanism dependent on structural remodeling and interdomain crosstalk. Biophysical methods have probed the impact of allosteric perturbations on cleavage and specificity of Cas9, with the aim of engineering enhanced Cas effectors. These studies include Cas9 from thermophilic organisms that edit at higher temperatures and are active in human plasma. Validation of biophysical insights has necessitated the quantitation of DNA cleavage in vitro and, subsequently, the adaptation of established protocols to encompass temperature-dependent function that is evident in extremophilic Cas systems, such as Cas9 from <i>Geobacillus stearothermophilus</i> and the mesophilic <i>Sp</i>Cas9. This protocol is advantageous for probing functional temperature ranges of DNA cleavage that can theoretically be applied to any Cas-RNP system. Key features • Builds upon the original Cas9 cleavage assays reported by Jinek et al. [1] to include the active temperature range of thermophiles. • Validated for assessing the cleavage activity of both mesophilic and thermophilic Cas9 systems. • Allows for qualitative and quantitative assessment of DNA cleavage across multiple physiological regimes. • Can be adapted to assess cleavage at multiple genomic loci or with different PAM requirements. • Assay can be completed in a single day.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5463"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514136/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282177","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 Agarose-Based FFPE Cancer Organoids for Morphology Preservation.","authors":"Mi Rim Lee, Sumin Kang, A-Ra Jeon, Sung-Woen Choi, Sun-Young Kong, Yun-Hee Kim","doi":"10.21769/BioProtoc.5467","DOIUrl":"10.21769/BioProtoc.5467","url":null,"abstract":"<p><p>Formalin-fixed paraffin-embedded (FFPE) slides are essential for histological and immunohistochemical analyses of organoids. Conventional preparation of FFPE slides from organoids embedded in basement membrane extract (BME) presents several challenges. During the fixation step, dehydration often causes collapse of the BME, which normally supports the three-dimensional architecture of organoids. As a result, organoids may lose their original morphology, particularly in the case of cystic or structurally delicate types, leading to distortion and reduced reliability in downstream histological evaluation. Here, we introduce a straightforward protocol that improves the reliability of FFPE slide preparation for BME-based organoids by enhancing sample integrity and sectioning quality. By using 2% agarose as a mold during the embedding process, organoids grown in BME were effectively stabilized, enabling reliable preservation of their morphology throughout FFPE slide preparation. This method effectively addresses the difficulties in processing structurally delicate organoids and allows robust preparation of diverse cancer organoid morphologies-such as cystic, dense, and grape-like structures-while maintaining their native three-dimensional architecture. Our approach simplified the technical process while ensuring reliable histopathological analysis, making it a valuable tool for cancer research and personalized medicine. Key features • FFPE preparation applicable to diverse cancer organoid morphologies, including cystic, dense, and grape-like, while preserving three-dimensional architecture. • Agarose molding allows intact retrieval and fixation of BME domes, preventing collapse and maintaining organoid 3D architecture during FFPE preparation. • Compatible with diagnostic IHC/IF markers (pan-CK, CK19, p63, Ki-67, p53) across cancer organoids.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5467"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514130/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282180","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":"Standardized Culture of Skin Fibroblasts From Punch Biopsies for Germline DNA Isolation in Myeloid Malignancies: A Practical Bedside-to-Laboratory Approach.","authors":"Parampreet Kour, Nedhi Kumari, Naveen Kaushal, Tanvi Sharma, Shano Naseem, Jogeshwar Binota, Manupdesh Singh Sachdeva, Arihant Jain, Manish Rohilla, Reena Das, Pankaj Malhotra, Pulkit Rastogi","doi":"10.21769/BioProtoc.5469","DOIUrl":"10.21769/BioProtoc.5469","url":null,"abstract":"<p><p>Inherited germline variants are now recognized as important contributors to hematologic myeloid malignancies, but their reliable detection depends on obtaining uncontaminated germline DNA. In solid tumors, peripheral blood remains free of tumor cells and therefore serves as a standard source for germline testing. In contrast, peripheral blood often contains neoplastic or clonally mutated cells in hematologic malignancies, making it impossible to distinguish somatic from germline variants. This unique challenge necessitates using an alternative, non-hematopoietic tissue source for accurate germline assessment in patients with hematologic myeloid malignancies. Cultured skin fibroblasts derived from punch biopsies have long been considered the gold standard for this purpose. Nevertheless, most existing protocols are optimized for research settings and lack detailed, patient-centric workflows for routine clinical use. Addressing this translational gap, we present a robust, enzyme-free protocol for culturing dermal fibroblasts from skin punch biopsies collected at the bedside during routine bone marrow procedures. The method details practical bedside collection, sterile transport, mechanical dissection without enzymatic digestion, plating strategy, culture expansion, and high-yield DNA isolation with validated purity. By integrating this standardized approach into routine hematopathology workflows, the protocol ensures reliable germline material with minimal patient discomfort and a turnaround time suitable for clinical diagnostics. Key features • This protocol integrates bedside skin punch biopsy with routine bone marrow sampling to minimize patient discomfort and avoid additional invasive procedures. • It uses an optimized enzyme-free mechanical dissection method with fat removal, fine mincing, and scratched well plating to reduce contamination and improve fibroblast yield. • It provides an easy-to-follow workflow for primary fibroblast culture, plating, expansion, and harvest, suitable for routine hematopathology laboratories. • It consistently yields high-quality germline DNA free of hematopoietic contamination, ideal for genetic testing in myeloid malignancies.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5469"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514132/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282112","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":"Artificial Metalloenzymes in Artificial Sanctuaries Through Liquid-Liquid Phase Separation.","authors":"Kaixin Wang, Guangjie Zhang, Lei Zhang, Yugang Bai, Tong Wu","doi":"10.21769/BioProtoc.5465","DOIUrl":"10.21769/BioProtoc.5465","url":null,"abstract":"<p><p>Artificial metalloenzymes (ArMs) hold great promise for expanding the toolbox of non-natural transformations usable in living systems, such as cells, plants, and animals. However, their practical application remains challenging, primarily due to their unsatisfactory stability and inefficient intracellular assembly. We recently reported a new strategy, called artificial metalloenzymes in artificial sanctuaries (ArMAS) through liquid-liquid phase separation (LLPS), to enhance the performance of ArMs in cells by placing them in more friendly artificial microenvironments. Here, this protocol describes the detailed method for using this ArMAS-LLPS strategy, a robust way to create artificial compartments using an ArM protein scaffold through LLPS and construct ArMs within using self-labeling cofactor anchoring reactions. In detail, in <i>Escherichia coli</i>, membraneless protein condensates are formed by expressing a self-labeling fusion protein, HaloTag-SNAPTag (HS) and act as intracellular sanctuaries. Simultaneously, the HS scaffolds enable site-specific, bioorthogonal conjugation with synthetic metal cofactors, facilitating efficient ArM formation within the LLPS domains. This strategy can significantly enhance the intracellular catalytic activity and stability of the named HS-based ArMs, allowing whole-cell catalysis to be performed to enable abiotic transformations both in vitro and in vivo. The protocol provides a proof-of-concept approach for researchers aiming to develop stable ArM-based whole-cell catalytic systems for synthetic biology and therapeutic applications. Key features • Describes a robust and reproducible protocol for constructing artificial metalloenzymes (ArMs) in living <i>E. coli</i> cells using protein-driven liquid-liquid phase separation (LLPS). • Demonstrates how intracellular LLPS regions can serve as protective catalytic microenvironments, significantly improving ArM stability and catalytic turnover. • Applicable to various abiotic catalytic transformations, including olefin metathesis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5465"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514139/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282152","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":"Detection of Plant RNA-Protein Interactions Using GFP-tag for Immunoprecipitation.","authors":"Fernanda Marchetti, Ayelen Distéfano, Gabriela Pagnussat, Eduardo Zabaleta","doi":"10.21769/BioProtoc.5466","DOIUrl":"10.21769/BioProtoc.5466","url":null,"abstract":"<p><p>The study of RNA metabolism involves understanding how RNA molecules interact with specific RNA-binding proteins (RBPs). In plants, these interactions have traditionally been investigated using a variety of in vivo and in vitro approaches, such as electrophoretic mobility shift assays or the analysis of knockout mutants. More recently, immunoprecipitation-based techniques have been developed. Most of the available protocols rely on crosslinking procedures, magnetic beads, and RNA-seq as the final endpoint analysis. Here, we present a protocol developed to identify specific RNA targets that directly interact with known plant RBPs using GFP-Trap^® agarose (ChromoTek) for immunoprecipitation without the need for crosslinking or RNA-seq. Briefly, a GFP-tagged RNA-binding protein is expressed in plant tissue, protein extracts are incubated with the GFP-Trap^® agarose matrix, and the resulting complexes are isolated. Co-purified RNAs, specifically mRNAs, are then analyzed by RT-PCR to detect bound transcripts. This protocol was first implemented for the study of RNA-protein interaction in <i>Arabidopsis thaliana</i>. This approach presents high potential for analysis in other plant species as well as several advantages, such as its high specificity and low cost. Even though GFP-Trap^® magnetic agarose (ChromoTek) has been used in plant systems to detect RNA-protein interactions, the protocol presented here consists of an alternative that is straightforward to implement when both candidate RNAs and RNA-binding proteins are known, and it can be broadly applied to study RNA-protein interactions in other plant systems. Key features • Can be used to confirm predicted RNA-protein interactions. • Suitable for validating RNA-protein interactions when candidate transcripts and RBPs are already known. • Compatible with downstream analysis by RT-PCR; can be adapted to RNA-seq if high-throughput data is needed. • Does not require crosslinking or specialized equipment beyond standard molecular biology tools for direct and strong RNA-protein interactions.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5466"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514128/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282203","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":"Fluorescence Lifetime-Based Separation of FAST-Labeled Cellular Compartment.","authors":"Aidar R Gilvanov, Ilya D Solovyev, Alexander P Savitsky, Mikhail S Baranov, Yulia A Bogdanova","doi":"10.21769/BioProtoc.5460","DOIUrl":"10.21769/BioProtoc.5460","url":null,"abstract":"<p><p>Here, we present a protocol for implementing the fluorogen-activating protein FAST (fluorescence-activating and absorption-shifting tag) in fluorescence lifetime imaging microscopy (FLIM), which allows separating fluorescent species in the same spectral channel based on fluorescence lifetime properties. Previous studies have demonstrated FLIM multiplexing using various combinations of synthetic probes, fluorescent proteins, or self-labeling tags. In this protocol, we utilize engineered FAST point mutation variants that bind fluorogen HBR-2,5-DM. The designed probes possess nearly identical, compact protein sizes (14 kDa), and the resulting protein-fluorogen complexes demonstrate comparable steady-state optical properties and exhibit distinct fluorescence lifetimes, displaying monoexponential fluorescence decay kinetics. When FAST variants are expressed with localization signals, these properties facilitate robust signal separation in regions with co-localized or spatially overlapping labels (nucleus and cytoskeleton in this protocol) in live mammalian cells. This method can be applied to separate other overlapping cellular compartments, such as the nucleus and Golgi apparatus, or mitochondria and cytoskeleton. Key features • The protocol employs FAST protein technology for fluorescent labeling. • Separation of cellular compartments in the green channel (emission wavelength ~500-550 nm) using fluorescence lifetime data. • Requires no coding skills. • The protocol is optimized for SPCImage software.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5460"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514129/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282134","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 Protocol Guide to Micro Milling for Bio-Microfluidics.","authors":"Hannah L Viola, Vishwa Vasani, Shuichi Takayama","doi":"10.21769/BioProtoc.5459","DOIUrl":"10.21769/BioProtoc.5459","url":null,"abstract":"<p><p>Micro milling is a subtractive manufacturing method for fabricating micro-scale three-dimensional features from hard substrates like acrylic, wood, or metal. It enables rapid prototyping of biomicrofluidic devices and master molds, offering advantages over traditional fabrication methods like photolithography. Micro milling is seldom applied in the fabrication of organs-on-a-chip, in part due to its requirement for knowledge of computer numerical machining techniques that are required to program and operate micro mills. This protocol provides practical guidelines for micro milling-based fabrication of organs-on-a-chip, including toolpath optimization, SolidWorks and Fusion workflows, and troubleshooting tips. A case study demonstrates the design and fabrication of master molds for a human airway-on-a-chip, validated in a recent publication. This resource supports the expansion of micro milling techniques into organs-on-a-chip, which will enhance capacity for rapid device prototyping and design of more complex 3D features that better recapitulate human physiology. Key features • Stepwise guide to lung-on-a-chip design and fabrication via micro milling, a specialized type of computer numerical control (CNC) machining. • Demonstration of model design, tool path optimization, micro milling, device assembly, and cell culture. • Example SolidWorks and Fusion documents illustrating best practices for model design and toolpath generation.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 19","pages":"e5459"},"PeriodicalIF":1.1,"publicationDate":"2025-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12514131/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145282139","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}