Bio-protocol最新文献

{"title":"Quantifying Epigenetic Changes Induced by Chemical Exposure Using the epi-TK Assay.","authors":"Shiro Kuroki, Haruto Yamada, Mizuki Odagiri, Kei-Ichi Sugiyama, Manabu Yasui, Akira Sassa","doi":"10.21769/BioProtoc.5671","DOIUrl":"https://doi.org/10.21769/BioProtoc.5671","url":null,"abstract":"<p><p>Epigenetic modifications play essential roles in regulating gene expression and maintaining cellular identity. Accumulating evidence suggests that chemical agents can contribute to carcinogenesis through epigenetic alterations, such as changes in DNA methylation and histone modifications, even in the absence of direct DNA damage. Here, we have developed a simple, cost-effective, and quantitative reporter assay, termed the epi-TK assay, to evaluate chemically induced epigenetic alterations. The assay is built upon the thymidine kinase (<i>TK</i>) gene mutation assay, a standardized and widely used in vitro genotoxicity assay for chemical safety evaluation. This system is based on an engineered human lymphoblastoid cell line (mTK6), in which the promoter region of the endogenous housekeeping <i>TK</i> gene is site-specifically methylated using epigenome-editing technology, resulting in stable transcriptional repression. Following chemical exposure, epigenetic perturbations at the <i>TK</i> locus are detected by culturing cells under hypoxanthine-aminopterin-thymidine selection and quantifying the frequency of TK revertant colonies, which reflects restoration of <i>TK</i> gene expression. Using the DNA methyltransferase 1 inhibitor GSK3484862 as a model compound, this protocol demonstrates that the epi-TK assay enables sensitive and quantitative detection of epigenetic state transitions. Importantly, this assay allows bi-directional detection of epigenetic changes, including DNA demethylation events and broader alterations in histone modification landscapes. Together, the epi-TK assay provides a practical and quantitative platform for evaluating epigenetic toxicity, with potential applications in chemical safety assessment frameworks. Key features • This protocol describes testing of the epigenetic effects of chemicals using the mTK6 cell line and a modified version of the <i>TK</i> gene-mutation assay. • By employing a DNA-methylated housekeeping <i>TK</i> gene and colony formation as the readout, the assay enables quantitative epigenetic changes without the need for specialized equipment. • The protocol offers a simple, quantitative, and cost-effective platform that is suitable for routine testing and comparative assessment of multiple compounds.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5671"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103964/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792077","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 Step-by-Step Protocol for the Isolation of <i>Aloe vera</i>-Derived Extracellular Vesicles via Manual and Shear-Force Homogenization.","authors":"Camila M Ceballos-Santa, Isabella Sanchez-Ortiz, Thomas R Gaborski, Karin Wuertz-Kozak","doi":"10.21769/BioProtoc.5664","DOIUrl":"https://doi.org/10.21769/BioProtoc.5664","url":null,"abstract":"<p><p><i>Aloe vera</i> has long been used for its diverse pharmacological properties, motivating continued interest in isolating and preserving the bioactive molecules responsible for its therapeutic potential. More recently, <i>Aloe vera</i>-derived extracellular vesicles (Av-EVs) have emerged as nanoscale, cell-free carriers capable of retaining and delivering these properties, making them attractive for various biomaterials, nanomedicine, and regenerative medicine applications. Multiple techniques are available for extracellular vesicle isolation. These include ultracentrifugation, polymer-based precipitation, size-exclusion chromatography, immunoaffinity capture, ultrafiltration, density gradient separation, and emerging microfluidic platforms. Each method presents distinct trade-offs in purity, yield, scalability, and downstream compatibility. Despite this diversity, standardized workflows tailored to Av-EV isolation remain limited, and the influence of homogenization-induced shear forces and plant maturity on vesicle recovery and characterization has not been systematically addressed. Here, we present a reproducible protocol for isolating Av-EVs from <i>Aloe vera</i> gel employing two distinct homogenization strategies: manual, no-shear force (NB EVs), and blender-based shear-force homogenization (B EVs). The workflow covers gel preparation, serial centrifugation for debris removal, ultracentrifugation as the gold standard for vesicle enrichment, and final sterile filtration. This protocol enables consistent recovery of Av-EVs suitable for physicochemical characterization and functional analyses. It is simple and relies on commonly available laboratory equipment, facilitating broad adoption by ultracentrifugation users and offering adaptability to diverse research projects involving purified <i>Aloe vera</i> gel and Av-EVs, including studies focused on wound healing, fibrotic scarring, and regenerative processes, where coordinated antioxidant, anti-inflammatory, antimicrobial, immunomodulatory, and moisturizing responses are of interest. Key features • This protocol allows direct comparison of vesicle yield, size distribution, and protein content across extraction methods. • This protocol yields ~1.4-2.0 × 10¹⁰ particles/mL per mature leaf for a total of ~8 × 10¹² particles per leaf. • This protocol yields ~1.2-2.8 × 10¹⁰ particles/mL per young leaf for a total of ~2.8 × 10¹² per leaf. • EVs from mature <i>Aloe</i> leaves yield protein concentrations of ~160-447 μg/mL, corresponding to ~3,840-10,728 μg of protein per leaf.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5664"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103970/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792117","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":"Protocol for Using CRISPR-Cas9 to Generate a Monocyte Cell Line Harboring a Single-Nucleotide Polymorphism.","authors":"Amar Chandra Mahatha, Octavio Ramos-Espinosa, Dhemerson Souza de Lima, Ethan Y Liu, Sarah Vaidyan, Jianguo Liu","doi":"10.21769/BioProtoc.5660","DOIUrl":"https://doi.org/10.21769/BioProtoc.5660","url":null,"abstract":"<p><p>We established a step-by-step approach for generating a single-nucleotide mutation in the promoter region of an immune regulatory gene in human monocyte THP-1 cells by employing a plasmid-based CRISPR-Cas9 system delivered via transfection with a homology-directed repair template DNA (HDR). Key steps include designing a single-guide RNA (sgRNA), cloning it into a CRISPR plasmid encoding the Cas9 protein, transfection of the plasmid constructs along with single-stranded oligonucleotide repair template (ssODNs) into THP-1 cells, followed by selection and validation. This approach provides a precise and relevant model to investigate the role of single polymorphisms in the regulation of inflammatory gene expression in human monocytes. In addition to the rs1024611 single-nucleotide polymorphism (SNP), this CRISPR/Cas9-based strategy is broadly applicable to functional studies of noncoding and coding variants in innate immune genes. Key features • Precise genome editing: CRISPR-Cas9-mediated editing of the rs1024611 SNP in the endogenous <i>CCL2</i> promoter in THP-1 cells using a single-stranded donor oligonucleotide. • Generate THP-1 monocytic cell lines: Differing only at the rs1024611 locus, allowing precise comparison of allele-specific effects on the same genetic background. • The protocol includes strategies for single-cell cloning, PCR-based genotyping, and Sanger sequencing to confirm precise genome editing. • While this study focuses on the <i>CCL2</i>-2518 SNP, it provides a framework for future investigations of other noncoding variants or regulatory SNPs in immune-related genes.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5660"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103971/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792134","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 Design to Practice: A Comprehensive Tutorial for the Rapid Multiplex Engineering of <i>Escherichia coli</i> Using Antibiotic Resistance Markers.","authors":"Shubhika Munot, Shuai Li, Jennifer N Hennigan, Eirik A Moreb, Michael D Lynch, Romel Menacho-Melgar","doi":"10.21769/BioProtoc.5665","DOIUrl":"https://doi.org/10.21769/BioProtoc.5665","url":null,"abstract":"<p><p>Engineering of microbial cells, including <i>E. coli</i>, is essential in prototyping genetic designs used in numerous applications throughout synthetic biology. While many advanced genome editing tools, such as CRISPR-based tools, offer new capabilities with genetically recalcitrant organisms, these tools often do not offer an immediate advantage in readily manipulated microbes, such as <i>E. coli</i>, especially when scarless modifications are not critical. We describe a comprehensive recombineering tutorial that we commonly use for multiplex engineering of <i>E. coli</i> using antibiotic markers. We leverage a group of 15 antibiotic resistance cassettes, most of which can be readily included when designing double-stranded DNA donors intended for recombineering and purchased from several vendors. Using these methods, 10-15 defined modifications to a single host strain can be achieved in less than three weeks, using two-day editing cycles. We discuss sequences and protocols as well as the optimal design of genetic modifications and the associated DNA. Key features • Enables 10-15 genomic modifications in less than 3 weeks. • Includes a comprehensive tutorial on designing donor DNA as well as choosing the appropriate antibiotic markers. • Includes DNA sequences that are amenable to commercial DNA synthesis.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5665"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103967/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147791673","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":"Workflow for Crystallographic Fragment Screening by Crystal Soaking for Protein Targets: A Case Study on Thioredoxin Glutathione Reductase From <i>Schistosoma mansoni</i>.","authors":"Lauro R S Neto, Bogar O Montoya, José Brandão-Neto, Kelly Parker, Frank Von Delft, Nicholas Furnham, Raymond J Owens, Floriano P Silva-Jr","doi":"10.21769/BioProtoc.5651","DOIUrl":"https://doi.org/10.21769/BioProtoc.5651","url":null,"abstract":"<p><p>Among the biophysical techniques used in fragment-based drug discovery (FBDD) campaigns, crystallography is the most sensitive, allowing for the identification of low-affinity ligands and the characterization of protein-ligand complexes at atomic resolution. Although powerful, the proper application of this technique depends on obtaining crystals capable of diffracting X-rays at high resolution. Additionally, in crystallographic compound screening, the crystals must be resistant to multiple organic solvents used in chemical libraries, such as DMSO. In this protocol, we describe recombinant protein production suitable for crystallization and procedures for X-ray crystallographic screening of a library of 768 fragments. As a case study, we used the <i>Schistosoma mansoni</i> thioredoxin glutathione reductase (SmTGR), a redox enzyme with a key role in controlling oxidative stress in parasites of the <i>Schistosoma</i> genus, which causes schistosomiasis. As a validated pharmacological target, SmTGR is used in the development of new schistosomicidal drugs. The experimental pipeline includes SmTGR expression, purification, and crystallization, crystal soaking, diffraction data collection, and refinement. The 768 fragments from the Diamond-SGC Poised Library (DSPL) were individually soaked onto the crystals, and diffraction data were collected and processed at the I04-1 beamline of the Diamond Light Source synchrotron. Diffraction data were subsequently analyzed using PanDDA to identify fragment-binding events and to enable reliable detection of low-occupancy ligands within the protein crystal structures. In addition to the core experimental steps, this protocol incorporates systematic approaches to overcome limitations frequently encountered in crystallographic screening campaigns, including assessment of crystal solvent tolerance, acceleration of crystal mounting through the use of auxiliary devices, acoustic dispensing-based soaking of hundreds of fragments for low material consumption and high throughput, automated data collection, and efficient data analysis pipeline for the detection of weakly bound ligand. This protocol can be broadly applied to screen diverse compound sets against multiple targets amenable to crystallization. Key features • Obtaining SmTGR through expression in ExpiSf9 cells with proper yield and purity for crystallization assays. • Systematic testing of buffer solutions to determine crystallization conditions, assessment of crystal tolerance to DMSO, and crystallographic data collection. • Integration of crystallization, acoustic dispensing, shifter-aided crystal mounting, data collection, and analysis powered by an in-house software pipeline. • This protocol builds upon the method developed by [1] and extends its application to other soluble proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5651"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103748/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792105","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 Control of Actin Network Assembly on the Supported Lipid Bilayer.","authors":"Kei Yamamoto, Makito Miyazaki","doi":"10.21769/BioProtoc.5656","DOIUrl":"https://doi.org/10.21769/BioProtoc.5656","url":null,"abstract":"<p><p>The spatiotemporal dynamics and density of actin networks are key determinants of actin cytoskeleton-mediated cellular functions. In vitro reconstitution systems have been widely used to study actin cytoskeletal dynamics; however, many existing approaches offer limited flexibility in controlling the geometry, thickness, and density of the assembled actin networks. Here, we present an in vitro optogenetic protocol that enables precise control of actin network assembly on supported lipid bilayers using an improved light-induced dimer (iLID)-SspB-based light-inducible dimerization system. In this system, His-mEGFP-iLID is anchored to a Ni-NTA-containing lipid bilayer, while SspB-mScarlet-I-VCA, a nucleation-promoting factor fused with SspB, together with other actin cytoskeletal proteins, is supplied in bulk solution. Upon blue light illumination, SspB-mScarlet-I-VCA is recruited to the membrane in a spatially and temporally defined manner, inducing localized actin polymerization. By tuning illumination patterns and duration, actin networks with defined density, thickness, and geometry can be generated, and polymerization can be rapidly halted by stopping illumination. This protocol provides a versatile platform for reconstructing actin networks with controlled spatial organization and density, enabling quantitative analysis of density-dependent interactions between actin networks and actin-binding proteins. Key features • Actin networks with varying densities and arbitrary shapes can be formed on the same supported lipid bilayer by controlling blue light illumination through the objective lens. • Actin polymerization can be stopped simply by turning off blue light illumination, enabling the formation of actin networks with defined thicknesses. • This protocol requires purified actin and actin-binding proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5656"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103897/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147791806","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":"Manipulation of Gene Expression in Mouse Pancreas via Intraductal Delivery of Adeno-Associated Viral Vectors.","authors":"Galina Soukhatcheva, Liam Stanley, Lei Dai, Mitsuhiro Komba, Volodymyr Andriiets, James D Johnson, Bruce C Verchere, Yi-Chun Chen","doi":"10.21769/BioProtoc.5657","DOIUrl":"https://doi.org/10.21769/BioProtoc.5657","url":null,"abstract":"<p><p>The rising global incidence of pancreatitis, pancreatic cancer, and diabetes has increased the need for efficient in vivo gene manipulation approaches to study the pancreas and develop new therapies. Although transgenic mouse models are widely used, they are time-consuming and costly to generate and maintain. Systemic viral delivery methods offer greater flexibility but often lack pancreatic specificity and require high viral doses. Here, we describe a streamlined protocol for intrapancreatic ductal delivery of adeno-associated viruses (AAVs) for targeted gene delivery. Our protocol requires standard surgical equipment and can be implemented in most laboratories. Specifically, we adopted a clamping strategy at the hepatopancreatic duct near the liver, as well as beneath the major duodenal papilla at the duodenum. This strategy exposes the duodenal papilla, facilitating viral delivery, preventing backflow, and enabling efficient pancreatic transduction at lower viral doses. Overall, this method provides a fast, simple, and effective approach for pancreas-targeted gene manipulation, facilitating preclinical studies of pancreatic biology and disease. Key features • Rapid, pancreas-specific in vivo gene manipulation using simple rodent surgical techniques. • Efficient gene manipulation can be achieved with lower viral doses while minimizing off-target effects. • AAVs trigger minimal adverse complications, and the surgery is well-tolerated in mice. • This method can be combined with traditional genetic manipulation and lineage tracing to enhance studies of gene function or pancreatic diseases.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5657"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103899/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147791846","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-Based Ion Transport Assays Using Proteoliposomes.","authors":"Karthik Ramanadane, Elena F Lehmann, Cristina Manatschal","doi":"10.21769/BioProtoc.5679","DOIUrl":"https://doi.org/10.21769/BioProtoc.5679","url":null,"abstract":"<p><p>Divalent metal ion transporters are conserved across all domains of life and play essential roles in diverse processes such as manganese acquisition during nutritional immunity in bacteria and iron homeostasis in higher eukaryotes [1-3]. Traditional techniques, such as electrophysiological assays, are often unsuitable due to the slow kinetics of many membrane transporters, electroneutral nature of certain transporter types, and the influence of other proteins with similar activity. To overcome these limitations and to investigate both the activity and ion selectivity of transporters, also including those normally expressed intracellularly, we have developed a fluorescence-based transport assay using purified proteins. This in vitro assay uses encapsulated fluorophores to monitor the movement of divalent metal ions (e.g., Mn²⁺, Ca²⁺, Mg²⁺) or protons across liposomal membranes reconstituted with purified transporter proteins. This approach provides detailed functional insight that complements structural and cellular data. Key features • Enables detection of real-time transport activity through precise timing of reagent addition and controlled generation of membrane potential. • Compatible with a wide range of divalent metal ions and ionophores, allowing adaptation to various transporter types. • Applicable to transporters that are naturally expressed in intracellular compartments, but requires a purified protein sample. • Allows detailed analysis of transporter function in a defined lipid environment and testing effects of binders and compounds.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5679"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103965/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792085","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 Splenic Natural Killer Cells of Mice Using Imaging Flow Cytometry.","authors":"Mohammad N Amin, Md Saqline Mostaq, Mohammad B Uddin, Yong-Yu Liu","doi":"10.21769/BioProtoc.5666","DOIUrl":"https://doi.org/10.21769/BioProtoc.5666","url":null,"abstract":"<p><p>Natural killer (NK) cells are crucial innate immune effectors, mediating cytotoxicity against cancer and infected cells through receptors such as NKG2D. Reliable quantification of NK cell subsets is essential for evaluating NK cell-based immune responses in cancer research. Unlike other assays, including traditional flow cytometry used in assessing NK cells, imaging flow cytometry (IFC) is a simple and direct method for quantitative analysis of NK cells. This protocol describes the necessary procedures, including harvesting splenocytes, acquiring these cells labeled with NKG2D antibodies, and analyzing IFC data with IDEAS^® software. We applied this protocol to quantitatively assess the number of splenic NKG2D⁺ NK cells in mice injected with SVTneg2 cancer cells (which carry the p53 G242A missense mutation) and compared them to mice injected with EMT6 cancer cells (which have wild-type p53) or normal fibroblasts. We found that the SVTneg2 cancer cells significantly decreased the number of NKG2D⁺ NK cells in mice by approximately 2-fold (933 cells vs. 2360 cells, p < 0.001) compared with mice injected with EMT6 cancer cells. This IFC protocol can be applied to directly quantify NK cells in vivo. This quantitative protocol allows novices to quickly handle the analysis of cytotoxic NK cells with a single NKG2D marker. Further multicolor flow cytometry and cytokine assay may be required to precisely define the subtypes and effects of NK cells in anticancer immunity. Key features • A simple and direct assay using imaging flow cytometry (IFC) to quantify cytotoxic NKG2D NK cells against breast cancer cells in mice. • Simultaneously collect the flow cytometry characters and each cell image of NK cells and other populations. • Step-by-step identification of interested NK cells mainly relying on image gating (focus, size, morphology). • Highly reliable and applicable to analyze other immune cell subsets or tumor-associated populations with corresponding conjugate antibodies.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5666"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103969/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792095","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 Feeder Cell-Free System for Chimeric Antigen Receptor Gene Transduction Into Natural Killer Cells.","authors":"Nobuhiro Kubo, Minori Baba, Yuko Suzuki, Yasushi Kasahara, Ryosuke Hosokai, Masaru Imamura, Akihiko Saitoh, Chihaya Imai","doi":"10.21769/BioProtoc.5669","DOIUrl":"https://doi.org/10.21769/BioProtoc.5669","url":null,"abstract":"<p><p>Anti-CD19 chimeric antigen receptor (CAR)-natural killer (NK) cells are expected to demonstrate anti-CD19 CAR-T-cell-like efficacy against relapsed and refractory B-cell malignancies and autoimmune diseases, with fewer adverse events and the added advantage of permitting the use of allogeneic cells. However, the methodology for generating CAR-NK cells remains under development. Although various cell sources and expansion methods are available, feeder cells derived from cancerous tissue have been most commonly employed to promote ex vivo expansion of NK cells. In the protocol described herein, NK cells are expanded from adult peripheral blood mononuclear cells using CD2- and NKp46-specific stimulating antibodies in combination with multiple cytokines. The activated NK cells can be genetically modified using a retroviral vector. Subsequent culture of these cells yields large numbers of anti-CD19 CAR-NK cells. The current method, which enables feeder-free, large-scale generation of anti-CD19 CAR-NK cells, eliminates the risk of tumor cell contamination and may facilitate safer clinical application. Key features • This method for expanding human primary NK cells ex vivo uses stimulatory antibodies and multiple cytokines, without requiring feeder cells, usually derived from cancerous tissue. • NK cells are selectively expanded from unsorted peripheral blood mononuclear cells. • Retroviral vector efficiently mediates gene transfer into NK cells stimulated with the current method. • Although the cells were not sorted, gene transfer into T cells is minimal.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"16 8","pages":"e5669"},"PeriodicalIF":1.1,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13103968/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147792123","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}