Bio-protocol最新文献

{"title":"Engineering Decellularized Extracellular Matrix-Incorporated Apical-Out Airway Organoids.","authors":"Zhuowei Gong, Chika S Ikpechukwu, Dhruv Bhattaram, Amy L Ryan, Daniel J Weiss, Xi Ren","doi":"10.21769/BioProtoc.5799","DOIUrl":"10.21769/BioProtoc.5799","url":null,"abstract":"<p><p>The airway epithelium interfaces with the external environment through its apical surface and with the extracellular matrix (ECM) through its basolateral surface. To model this organization in vitro, we developed a decellularized ECM-incorporated apical-out airway organoid (dECM-AoAO) platform in which human bronchial epithelial cells (HBECs) self-assemble around human lung-derived decellularized ECM microparticles (dECM-MPs). This configuration preserves apical-out polarity while enabling direct epithelial-ECM interactions. Here, we describe a protocol for the vacuum filtration and quantification of dECM-MPs, the generation of dECM-AoAOs, and ultimately, whole-mount immunofluorescence staining for organoid characterization. Key features • This protocol incorporates dECM as size-refined microparticles, enabling a consistent and reproducible ECM input. • The workflow uses suspension culture to generate apicalout airway organoids, eliminating the need for matrix embedding while preserving ECM-cell interactions. • dECM-MPs are directly wrapped by epithelial cells, simplifying handling and supporting native-like epithelial-ECM integration. • The system is compatible with wholemount immunofluorescence staining for comprehensive epithelial lineage analysis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5799"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507971/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835715","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":"Optimized Buffer for Preservation of Hepatitis E Virus During Freeze-Thaw Cycles.","authors":"Zhe Jiao, Zongdi Feng","doi":"10.21769/BioProtoc.5781","DOIUrl":"10.21769/BioProtoc.5781","url":null,"abstract":"<p><p>Hepatitis E virus (HEV) is a zoonotic pathogen responsible for approximately 20 million infections annually worldwide. The lack of robust cell culture systems and the absence of approved antiviral therapies have hindered HEV research and drug development. A major technical challenge is the rapid loss of viral infectivity during freeze-thaw cycles following virus purification. Here, we describe a simple and reproducible method to preserve HEV infectivity during storage. We systematically evaluated the effects of salt, serum, and sucrose on viral stability under freezing conditions. We identified an optimized buffer containing 2% fetal bovine serum (FBS), 150 mM NaCl, and 7% sucrose, which significantly maintained the infectivity of non-enveloped HEV (nHEV) and quasi-enveloped HEV (eHEV) following freeze-thaw cycles based on immunofluorescence. The buffer also demonstrated good stability across three independent repeat infection experiments. This protocol provides a practical and scalable approach for maintaining HEV infectivity and will facilitate HEV-related virological studies. Key features • Optimized buffer formulation for preserving HEV infectivity during freeze-thaw cycles. • Compatibility with nHEV and eHEV. • Simple and low-cost formulation using FBS, NaCl, and sucrose. • Downstream validation using an infectivity assay.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5781"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507970/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835121","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":"Analysis of Bacterial-Mediated c-di-AMP Degradation by Thin-Layer Chromatography.","authors":"Anju Bala, Adriana G Morales Rivera, José A Lemos","doi":"10.21769/BioProtoc.5796","DOIUrl":"10.21769/BioProtoc.5796","url":null,"abstract":"<p><p>Cyclic di-AMP is a bacterial second messenger nucleotide required for the regulation of numerous cellular functions, including potassium and osmolyte homeostasis, DNA repair, cell wall integrity, central metabolism, and stress adaptation. This second messenger is synthesized from two ATP molecules by diadenylate cyclases (DAC) and degraded by cytoplasmic and surface-associated phosphodiesterases (PDE) to phosphoadenylyl adenosine (5' pApA), adenosine monophosphate (AMP), and, in some instances, adenosine and inorganic phosphate (Pi). Levels of c-di-AMP in bacteria can be determined using different methods, including liquid chromatography-mass spectrometry (LC-MS/MS), enzyme-linked immunosorbent assay (ELISA), and luminescent and fluorescent biosensors. Thin-layer chromatography (TLC) is another method routinely used to monitor c-di-AMP synthesis and degradation by purified DAC and PDE enzymes and is particularly useful for monitoring c-di-AMP degradation products. Here, we devised a TLC-based method to monitor extracellular c-di-AMP stability and degradation by intact bacterial cells using radiolabeled c-di-AMP. We show that bacterial strains of <i>Enterococcus faecalis</i> and <i>Streptococcus agalactiae</i> that possess surface-associated PDEs can rapidly degrade extracellular c-di-AMP. In addition, we demonstrate that this method can be used to indirectly identify alternative enzyme substrates through competition assays. We propose that this TLC-based assay is an efficient method to analyze bacterial-mediated degradation of c-di-AMP and is amenable to testing other radiolabeled nucleotides. Key features • This method makes use of TLC, a well-established technique, to visualize c-di-AMP degradation products. • Bypasses the need to obtain purified protein and instead uses live bacterial cells to monitor extracellular substrate degradation at the cell surface interface. • This protocol can be adapted to other organisms and cell types and to detect other radiolabeled substrates.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5796"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507967/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835658","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":"Clonal Analysis in <i>Drosophila</i> Tissues With an Enhanced MAGIC Transgenesis Method.","authors":"Yifan Shen, Chun Han","doi":"10.21769/BioProtoc.5787","DOIUrl":"10.21769/BioProtoc.5787","url":null,"abstract":"<p><p>Mosaic animals are highly valuable for investigating complex biological processes and cell lineages in vivo. Traditional mosaic techniques in <i>Drosophila</i>, such as the FRT/Flp system, rely on exogenous site-specific recombination sequences, preventing their application to unmodified mutant chromosomes or wild-derived strains. Mosaic analysis by gRNA-induced crossing-over (MAGIC) overcomes this limitation by utilizing the CRISPR/Cas9 system to generate targeted double-strand breaks (DSBs) that induce somatic homologous recombination in precursor cells. Here, we describe a comprehensive protocol for applying MAGIC with a newly developed, genome-wide MAGIC kit. This protocol utilizes optimized gRNA-markers with the Qtg2.1 scaffold for high-efficiency clone induction, alongside improved fluorescent labeling strategies for both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). The procedure details the genetic crossing schemes, temporal induction of clones, and tissue processing for diverse <i>Drosophila</i> cell types. This method enables convenient mosaic analysis across all chromosomes and allows for the study of pericentromeric genes, deficiency chromosomes, and species-specific alleles in interspecific hybrids. Key features • <b>Recombinase-independent:</b> Generates somatic mosaic clones using CRISPR/Cas9 without requiring pre-inserted FRT sequences on the test chromosome. • <b>Genome-wide application:</b> Includes a complete toolkit of pMAGIC and nMAGIC gRNA-markers optimized for all <i>Drosophila</i> chromosomal arms (X, 2L, 2R, 3L, 3R, and 4). • <b>Optimized labeling:</b> Employs destabilized Gal80 for brighter pMAGIC clones and <i>tub-3xHA-BFP/IFP</i> for unambiguous visualization of nMAGIC clones. • <b>Broad compatibility:</b> Applicable to a wide variety of tissues (e.g., neurons, glia, imaginal discs, polyploid tissues) and complex genetic backgrounds, including pericentromeric mutations and deficiencies.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5787"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507965/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835655","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 MATLAB-Based Image Processing Protocol for Quantitative Differentiation of Diseased and Healthy Plant Tissue From Digital Leaf Images.","authors":"Bhaskar Dowarah, Pankaj Borah, Rafiul Amin Laskar, Alinaj Yasin, Anurag Kashyap, Bijoy Neog","doi":"10.21769/BioProtoc.5798","DOIUrl":"10.21769/BioProtoc.5798","url":null,"abstract":"<p><p>Accurate quantification of plant disease severity is essential for evaluating host-pathogen interactions and assessing the effectiveness of disease management strategies. Traditional visual scoring methods and manual estimation of infected tissue are widely used but are often subjective and prone to observer bias. Digital image analysis offers an objective alternative by enabling automated identification and quantification of symptomatic plant tissues based on color and spatial characteristics. Here, we present a MATLAB-based image processing protocol for differentiating diseased and healthy plant tissue from digital leaf images. The workflow involves acquisition of standardized leaf images, conversion of RGB images into hue-saturation-value (HSV) color space, segmentation of diseased tissue using defined HSV thresholds, refinement of the segmented mask through morphological operations, and extraction of the whole leaf area. The protocol then calculates the diseased area and total leaf area in pixels and computes the percentage of infected tissue. The method uses MATLAB together with the Image Processing Toolbox and can be implemented using simple scripts. This protocol enables rapid and reproducible quantification of disease severity in plant leaves exhibiting visually distinct symptoms such as necrotic lesions or blight patches. By minimizing observer bias and providing quantitative measurements of infected area, the protocol offers a practical and reproducible approach for plant disease phenotyping and evaluation of disease management strategies across diverse plant-pathogen systems where diseased tissues can be clearly distinguished from healthy tissues under reasonably controlled imaging conditions. Key features • A reproducible MATLAB-based workflow for separating diseased and healthy plant tissue using color-space segmentation. • Applicable to plant diseases where symptomatic tissue contrasts clearly with healthy tissue (necrosis, blight lesions, rot patches). • Requires digital leaf images, MATLAB, and the MATLAB Image Processing Toolbox for image processing and disease quantification. • Enables rapid calculation of diseased leaf area and disease severity using automated pixel-based quantification.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5798"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507964/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835638","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":"Immersive Social Interaction Assay (ISIA) for Studying Voluntary and Long-Term Social Behavior in Mice.","authors":"Aaron Crane, Elizabeth Sorvino Mancini, Ada Eban-Rothschild","doi":"10.21769/BioProtoc.5789","DOIUrl":"10.21769/BioProtoc.5789","url":null,"abstract":"<p><p>Social behavior is highly dynamic and context-dependent, yet many commonly used rodent social assays rely on short testing periods and simplified measures of proximity or investigation. Here, we present a detailed protocol for constructing and using the immersive social interaction assay (ISIA), a behavioral paradigm designed to capture prolonged, voluntary social interactions in freely moving mice. The ISIA apparatus consists of two modified standard rodent home-cage chambers connected by a 3D-printed tube with an adjustable inner diameter. This design enables flexible experimental control: a removable restrictor can confine a head-bar-implanted focal mouse to one chamber while permitting a freely moving conspecific to traverse the full apparatus, enabling assessment of social motivation. Animals can be recorded over extended time windows with familiar or novel conspecifics, in their home territory or in novel environments, and under varying thermal or enrichment conditions, while expressing a broad repertoire of affiliative and aggressive behaviors, including huddling and aggression. The apparatus can be readily integrated with machine learning-based tracking and behavioral classification pipelines for high-throughput analysis. This protocol describes step-by-step construction, setup, and experimental implementation of the ISIA, from apparatus construction and head-bar implantation to video-based behavioral analysis, with the goal of facilitating broader adoption of ethologically relevant behavioral assays in neuroscience research. Key features • The ISIA apparatus is modular and cost-effective, built from standard rodent housing and 3D-printed components. • Enables voluntary, prolonged social interactions in mice, preserving the ability to engage or withdraw and allowing assessment of social motivation. • Provides flexible experimental control over access between animals (e.g., via an adjustable restrictor) while maintaining naturalistic interaction dynamics. • Integrates with machine learning-based tracking pipelines for automated, high-throughput quantification of complex social behaviors.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5789"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507975/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835700","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":"Identification of DNA-Binding Factor Enrichment in Chromatin Accessibility Data to Define a Persister Cell Signature.","authors":"Vidya Ajay, Mihai G Dumbrava, Alexandre Gaspar-Maia, Wazim Mohammed Ismail","doi":"10.21769/BioProtoc.5800","DOIUrl":"10.21769/BioProtoc.5800","url":null,"abstract":"<p><p>Chemotherapy-resistant persister cells are a major driver of cancer recurrence, yet their epigenetic basis remains poorly characterized. This protocol describes a computational pipeline for identifying DNA-binding factors (DBFs) that are enriched in accessible chromatin that collectively define a persister cell signature (PCS). Starting from single-nucleus ATAC-seq (snATAC-seq) data processed through the 10x Genomics CellRanger ARC pipeline, this protocol covers (1) the creation of a Seurat/Signac object with ATAC peaks, (2) the optional integration of DNA-binding data from the ReMap2022 database as a per-cell chromatin module assay, (3) differential accessibility analysis across clinically defined comparison groups, and (4) identifying and defining the top enriched DBFs as the PCS. This approach is applicable to any snATAC-seq dataset in which cells can be grouped by clinical response, treatment status, or resistance phenotype. Key features • Integrated analysis of chromatin accessibility data with publicly available DNA-binding data is a useful technique to identify potential epigenetic biomarkers. • Single-nucleus ATAC-seq from clinically relevant samples can be used to identify binding enrichment of specific DNA-binding factors in sub-populations of cells. • This protocol was used to identify a persister cell signature that was able to predict chemotherapy resistance in high-grade serous ovarian cancer.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5800"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507972/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835632","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":"Optical Tissue Clearing and Small-Molecule Labeling of Paraffin-Embedded Breast Cancer and Axillary Lymph Node Human Tissue Samples.","authors":"Lisa D J Schiffelers, Uma Pisarović, Albert Bitorina, Sven Hildebrand, Britt Walravens, Thiemo J A van Nijnatten, Loes F S Kooreman, Anna Schueth","doi":"10.21769/BioProtoc.5790","DOIUrl":"10.21769/BioProtoc.5790","url":null,"abstract":"<p><p>Breast cancer is the most frequently diagnosed cancer in women, representing approximately 25% of all cancers in women worldwide. Both breast cancer research and histopathological diagnostics mainly show a two-dimensional planar view of the three-dimensional breast cancerous architecture. Recently, the application of optical tissue clearing, together with 3D microscopy, has been applied to visualize the complexity of whole tumor samples. Preliminary studies on whole-organ mouse mammary glands and tissues from human breast cancer patients subjected to optical tissue clearing and volumetric imaging have enabled the detection of previously unrecognized spatial cellular interactions and structural features within intact breast tissue. There is currently no standardized clearing workflow for breast and lymph node tissues. In this protocol, we optimized and validated the MASH (multiscale architectonic staining of human cortex) immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO)-like clearing and labeling pipeline for the investigation of formalin-fixed and paraffin-embedded (FFPE) breast tissue and lymph nodes obtained from breast cancer patients. This illustrates the application of the protocol in a new biological and clinical context, as human breast and lymph node tissues differ substantially from brain tissues in their composition, architecture, and optical properties. Whole FFPE tissue blocks are deparaffinized in liquid paraffin and xylene, bleached through methanol dehydration and a subsequent hydrogen peroxide incubation, and stained with a diverse set of small molecule dyes. As a next step, the tissues are delipidated and subjected to refractive index matching with ethyl cinnamate to reach optimal tissue transparency. Importantly, the applied dehydration and delipidation nicely preserve the morphology of the tissue, and the shrinkage is minimal. This allows reliable 3D imaging of large tissue samples within a timeframe of 10 days, providing clinicians and biomedical researchers with a more holistic view of the FFPE tissue sample and its spatial organization. Key features • Optimized, low-cost approach for optical tissue clearing and labeling of whole human breast and lymph node FFPE tissue samples ≥30 × 20 × 3 mm. • Prepared tissue samples can be assessed by means of advanced 3D volume microscope modalities, such as twophoton microscopy (TPM) or light-sheet fluorescence microscopy (LSFM). • The clearing protocol is compatible with a variety of small-molecule dyes, such as neutral red (cell body), eosin Y (cytoplasm, collagen), and methyl green (nucleus).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5790"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507969/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835073","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":"Calcium Imaging in H4IIE Liver Cells and Primary Rodent Hepatocytes: A Cost-Effective Protocol for Use With Fura-2 AM Ca²⁺ Indicator.","authors":"Lu Fang, Ahmed G K Habib, Marlee D Harris, Alex J Begley, Eunus S Ali","doi":"10.21769/BioProtoc.5783","DOIUrl":"10.21769/BioProtoc.5783","url":null,"abstract":"<p><p>Calcium signaling is a universal, versatile process in which ionized or free calcium (Ca²⁺) acts as a second messenger to regulate various cellular activities, including hormone secretion, contraction, proliferation, gene expression, and apoptosis. Changes in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt) in hepatocytes play a central role in mediating the actions of insulin, glucagon, catecholamines, and other hormones on carbohydrate, lipid, and protein metabolism in the liver. Ratiometric chemical Ca²⁺ indicators are fluorescent dyes that change their emission or excitation spectrum upon binding to calcium, allowing for precise, quantitative measurements of changes in the intracellular Ca²⁺ concentration. They enable calibration by calculating the ratio of two fluorescence intensities, correcting for artifacts such as uneven dye loading, photobleaching, and cell volume variations. Fura-2 acetoxymethyl ester (AM) (hereinafter referred to as Fura-2), a ratiometric and sensitive indicator dye, is a popular fluorescent Ca²⁺ reporter for measuring intracellular calcium. Here, we describe a comprehensive and detailed protocol for Ca²⁺ imaging of the H4IIE cell line and primary rodent hepatocytes in vitro via the chemical reporter Fura-2, which can also be employed on a wide variety of cell types. Unlike previously published protocols, this protocol addresses the challenge of facilitating the attachment of liver cell lines and primary hepatocytes to glass coverslips for imaging using an inverted fluorescence microscope. Our protocol describes two different loading/labeling strategies for Fura-2 dye: one is cost-effective but requires skillful pipettor handling, and the second one is easy but expensive as it needs a large volume of Krebs-Ringer HEPES (KRH)-Fura-2 solution. If the coverslips are handled properly, the cost-effective coverslip-only loading approach produces similar quality results as the large volume method. Finally, we describe a simple and user-friendly procedure to analyze Ca²⁺ signals over time using Microsoft Excel's functional equations. Key features • This protocol enables real-time monitoring of intracellular Ca²⁺ in living hepatocytes. • The method is based on fluorescence microscopy of hepatocytes loaded with Fura-2 fluorescent dye; moreover, the protocol can be extended to other mammalian cell lines. • We used this protocol to monitor the activity of store-operated Ca²⁺ channels or SERCA pumps, but it could also be extended to other intracellular phenomena. • If the Fura-2 loading strategy and incubation time are followed properly, this protocol produces highly reproducible imaging results.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5783"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507977/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835675","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 Luciferase-Based Assay for Assessing Cap-Independent Translation in Wheat Germ Extract.","authors":"Max Cortot, Thorsten Stehlik, Aline Koch, Timo Schlemmer","doi":"10.21769/BioProtoc.5791","DOIUrl":"10.21769/BioProtoc.5791","url":null,"abstract":"<p><p>Efficient protein synthesis in eukaryotic cells typically requires a 5' cap structure on messenger RNAs (mRNAs). However, under stress conditions or in viral infection, translation can also occur independently of the cap via internal ribosomal entry sites (IRES). IRES elements are therefore key regulators of protein expression in both viral and cellular contexts. Here, we describe a cell-free protocol to quantitatively assess cap-independent translation using wheat germ extract (WGE) and a firefly luciferase (FLuc) reporter. The protocol includes template preparation, RNA synthesis, and luminescence measurement following in vitro translation in WGE. This method enables rapid and robust comparison of translation activity under controlled conditions and can additionally be applied to evaluate mRNA modifications designed to enhance translation efficiency. Key features • Stringent in vitro workflow from DNA template preparation through RNA synthesis and protein synthesis to reporter readout, including quality controls. • Evaluation of cap-independent translation suitable for testing combinations of IRES and CDS. • Translation analysis without radioactive labeling.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 16","pages":"e5791"},"PeriodicalIF":1.3,"publicationDate":"2026-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13507978/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148835722","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}