Jove-Journal of Visualized Experiments最新文献

{"title":"Preparation of Economical and Universal Compensation Beads Compatible for Multi-species Antibodies.","authors":"Shuanghe Peng, Linpei Guo, Di Yin, Zhun Wang","doi":"10.3791/70114","DOIUrl":"https://doi.org/10.3791/70114","url":null,"abstract":"<p><p>This protocol describes a cost-effective and reproducible method for preparing multi-species-compatible compensation beads for fluorescence compensation in multicolor flow cytometry. Here, recombinant the immunoglobulin binding protein A/G/L (Protein A/G/L) containing multiple immunoglobulin-binding domains was expressed in Escherichia coli (E. coli), purified using nickel affinity chromatography, and covalently coupled to carboxylated polystyrene microspheres through EDC/NHS chemistry. The resulting beads bind antibodies from diverse species and subclasses, generating strong and distinct fluorescence signals suitable for compensation setup. These results provide practical guidance for selecting bead diameter and protein loading to achieve fluorescence intensities appropriate for different compensation requirements. Successful execution of the protocol is indicated by efficient protein purification and a clear, high-intensity fluorescence peak of conjugated beads compared with control beads during flow cytometry analysis. This simple and scalable approach enables routine preparation of stable, versatile compensation beads in standard laboratories, reducing cost while improving flexibility for flow cytometry applications.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844994","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Three-Dimensional Co-Culture Method for the Study of Indirect Interactions Between Different Cell Types Across a Reconstituted Basement Membrane.","authors":"Grant R Kelly, Pere Miquel Morla-Barcelo, Nikitha K Pallegar, Jorge Sastre-Serra, Mercedes Nadal-Serrano, Alicia M Viloria-Petit, Sherri L Christian","doi":"10.3791/70027","DOIUrl":"https://doi.org/10.3791/70027","url":null,"abstract":"<p><p>Three-dimensional (3D) co-culture is a rapidly evolving technique for researchers looking to accurately study cell-cell interactions using in vitro experiments. The limitations of monolayer cell culture, including limited interactions between the cellular and extracellular environment and disturbed cell morphology, are addressed by simulating the in vivo cellular environment. Using a scaffold to provide structural support and including biologically active extracellular matrix components, 3D cultures display behaviours and morphologies more congruent with tissue. Incorporating multiple cell types into this kind of 3D environment allows for the study of cell-cell interactions inside a biomimetic model system. A wide range of 3D co-culture technologies has emerged, each with its own advantages and challenges. Often these technologies require specialized equipment, a complex setup, or specific technical knowledge. As well, there exist a few standardized methods for studying indirect cell-cell interactions between two cell types separated by a reconstituted basement membrane. Here, we describe a 3D co-culture method that requires only fundamental technical skills and uses more widely applicable materials to successfully recapitulate indirect cell-cell interactions across a basement membrane. A monolayer of cells is covered in a layer of extracellular matrix, in the form of Matrigel, and a second cell type is seeded on top. The resultant co-culture is maintained for five days, at which point cells are analyzed for morphological changes by immunofluorescence or extracted from the co-culture for more detailed genomic, transcriptomic, or proteomic analyses. This protocol is ideal for studying the impact of cell-cell communication on cell behaviour when physical contact is prohibited by a basement membrane. As researchers continue to opt for more in vivo-relevant cell culture methods, a streamlined approach is necessary to avoid high barriers to entry.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Building and Operating a Low-Cost Elevated Carbon Dioxide Growth Chamber to Evaluate Microgreen Physiology under Spaceflight-Relevant CO2 Levels.","authors":"T Aurora Toennisson, T Blake Horton, Samantha Rueckeis, Joe Chiera, Sam Cho, Imara Y Perera, Colleen J Doherty","doi":"10.3791/70050","DOIUrl":"https://doi.org/10.3791/70050","url":null,"abstract":"<p><p>Carbon dioxide (CO2) levels aboard crewed spacecraft routinely exceed 3,000 ppm, significantly higher than atmospheric concentrations on Earth. Sustained exposure to extreme CO2 (exCO2) at levels above 3,000 ppm can alter plant growth, physiology, and nutritional composition. Yet, few laboratory systems can reliably reproduce these exCO2 concentrations. This protocol describes an accessible and low-cost method for constructing a benchtop growth chamber capable of maintaining stable CO2 levels above 3,000 ppm for plant experiments. The chamber is created by modifying a benchtop incubator to include airtight cable and gas fittings, an automated CO2 delivery and venting system, and integrated temperature, humidity, and pressure sensors. The system is controlled by an inexpensive single-board computer, using open-source Python code to maintain a target CO2 concentration by regulating solenoids that inject CO2 or vent the chamber in response to system readings. A custom passive wicking box sustains plant growth without requiring active watering, reducing the need to open the chamber and minimizing fluctuations in CO2 levels. Using this system, we successfully grew two microgreen species, radish (Raphanus sativus, a C3 plant) and amaranth (Amaranthus cruentus, a C4 plant), under \"ambient CO2\" (minimum 400 ppm) and \"ISS-like exCO2\" (minimum 3,000 ppm) conditions. The chambers maintained stable CO2 levels, temperature, and humidity throughout the 10-14-day growth cycles. This reproducible, spaceflight-relevant CO2 system provides an accessible tool for studying plant responses to extreme atmospheric environments and for preparing experiments for spaceflight.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844835","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"New Islet Model for 3D Study of Endothelial and β Cell Interactions: Relevance for the Screening of Cytoprotective Agents.","authors":"Amira Saidi, Clémence Tollard, Liyao Liu, Yasmina El Khallouqi, Jean Sévigny, Cyril Auger, Gilles Kauffenstein, Fatiha El Ghazouani, Laurence Kessler, Florence Toti","doi":"10.3791/68220","DOIUrl":"https://doi.org/10.3791/68220","url":null,"abstract":"<p><p>This protocol details an optimized method for the production of small stable spheroids, their culture, and 3D imaging, for the study of the endothelial and insulin-producing β cells interactions in a 3D model of pancreatic islets. The 150-200 µm spheroids, mirroring the lowest range of islet sizes, were prepared from a selected ratio combining 1 intra-islet endothelial cells (MS-1 cells) to 20 insulin-secreting cells (β-TC-6). Staining, clearing, and mounting challenges of small spheroids and their tackling by employing low-melting point agarose and the CUBIC clearing technique are detailed, as well as key points for an efficient analysis of the 3D structure with different probes. Data indicate that NTPDASE-ectonucleotidase 3 does not colocalize with insulin in the spheroid model, suggesting varying maturity and functional levels of β-TC6 and that the complete procedure can also be applied to isolated pancreatic islets, with clear probing of intra-islet vessels. These findings underscore the effectiveness of the 3D imaging protocol in revealing complex pancreatic cell organization and interactions within the islet model.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844910","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Robotic Duodenum-preserving Total Pancreatic Head Resection for Intraductal Papillary Mucinous Neoplasms.","authors":"Mingfei Wang, Ming Cui, Menghua Dai","doi":"10.3791/69118","DOIUrl":"https://doi.org/10.3791/69118","url":null,"abstract":"<p><p>Robotic duodenum-preserving pancreatic head resection (R-DPPHR) is a technically feasible procedure for benign or borderline tumors of the pancreatic head. Among these, pancreatic cystic neoplasms (PCNs), particularly intraductal papillary mucinous neoplasms (IPMNs), are ideal candidates for this minimally invasive, organ-preserving approach. While numerous studies have demonstrated the short- and long-term advantages of DPPHR over conventional pancreaticoduodenectomy (PD), detailed video descriptions of the robotic-assisted procedure are scarce. This case report presents a step-by-step technical video of R-DPPHRt for a main duct-IPMN (MD-IPMN) in the pancreatic head. A 65-year-old male patient with upper abdominal discomfort was diagnosed with MD-IPMN, presenting as a 4.3 cm cystic-solid mass in the pancreatic head. R-DPPHRt was performed using the da Vinci Xi system, with an operation time of 350 minutes and an estimated blood loss of 150 mL. The patient had an uneventful postoperative course and was discharged on postoperative day 9. Pathology confirmed MD-IPMN with low-grade dysplasia. R-DPPHRt is an effective, function-preserving treatment for selected patients with pancreatic cystic neoplasms, particularly when performed by experienced surgeons in high-volume centers. While this single-case report limits generalizability, the article offers unique value by providing a clear procedural template.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combined Detection of IFN-γ And CD64 for Diagnosis and Monitoring of Active Pulmonary Tuberculosis in Elderly Patients.","authors":"Jing Zhou, Yuchun Cai, Dacheng Guo","doi":"10.3791/70389","DOIUrl":"https://doi.org/10.3791/70389","url":null,"abstract":"<p><p>This study assessed the risk of active pulmonary tuberculosis (ATB) in elderly patients with weakened immunity by jointly detecting serum interferon-γ (IFN-γ) and neutrophil CD64, and analyzed the impact of this diagnostic protocol on the course of ATB. A total of 50 patients with ATB and 50 age- and sex-matched healthy controls were recruited. Subsequently, the patient's neutrophil CD64 (quantified as the percentage of CD64-positive neutrophils [CD64%]), IFN-γ (by enzyme-linked immunosorbent assay [ELISA]), C-reactive protein (CRP; by immunoturbidimetry), and erythrocyte sedimentation rate (ESR; by Westergren method). Sputum samples from patients were cultured on Löwenstein-Jensen (L-J) medium and in the MGIT 960 automated system. The diagnostic efficacy of individual and combined biomarkers was assessed with receiver operating characteristic (ROC) curve analysis, and a logistic regression model was developed for combined detection. Significantly elevated levels of both IFN-γ and CD64 were observed in ATB patients versus healthy controls (P<0.05). A diagnostic model incorporating both biomarkers had an area under the curve (AUC) of 0.838, with a sensitivity of 84.00% and a specificity of 76.00%. Both markers decreased following therapeutic intervention (P<0.05), showing the lowest values in culture-negative patients (P<0.05). Furthermore, the combined model showed predictive utility for culture conversion, attaining an AUC of 0.756 (65.71% sensitivity, 80.00% specificity; P<0.001). The combined detection of IFN-γ and CD64 can effectively diagnose the occurrence of ATB in immunocompromised elderly people, providing a reference for clinical practice.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and Culture of White and Brown Preadipocytes from Mouse Embryos.","authors":"Martina Canatelli-Mallat, Mouna Amaouche, Silvia Bongiovanni, Alicia Mayeuf-Louchart","doi":"10.3791/70101","DOIUrl":"https://doi.org/10.3791/70101","url":null,"abstract":"<p><p>Adipose tissue plays a central role in metabolic homeostasis, and its properties are shaped during embryonic development through adipocyte differentiation. Embryonic preadipocytes, therefore, represent a relevant model to study early events in adipose tissue formation and lineage specification. This article describes a reproducible protocol for the isolation and in vitro differentiation of white and brown preadipocytes isolated from mouse embryos at embryonic day 15.5 (E15.5), a developmental stage at which adipose depots begin to form. The method provides detailed guidance for tissue microdissection, enzymatic digestion, primary culture, and lineage-specific differentiation conditions that support cell viability and adipogenic maturation. Representative results include lipid droplet accumulation and lineage-associated marker expression, confirming successful differentiation under defined culture conditions. Using this approach, embryonic preadipocytes can be directed toward white or brown adipocyte fates, enabling comparative analyses of developmental timing, lineage characteristics, and gene expression profiles. This protocol offers a practical and developmentally relevant tool for investigating adipose tissue formation and perinatal programming mechanisms in a controlled experimental setting.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrative Analysis of Targeted Genomic Profiling and Immune Cell Infiltration in the Prognosis of Lung Adenocarcinoma.","authors":"Xuechun Wang, Wei Luo, Xuehong Gu, Teng Hu, Guping Shi, Jia Jia, Hongmei Qiu, Yangfan Guo","doi":"10.3791/69843","DOIUrl":"https://doi.org/10.3791/69843","url":null,"abstract":"<p><p>The prognosis of lung adenocarcinoma (LUAD) is traditionally evaluated via tumor-node-metastasis (TNM) staging, which does not account for the patient's immune status. This protocol integrates genomic profiling and immune microenvironment analysis to facilitate a more comprehensive postoperative prognostic evaluation. The method involves a retrospective analysis of paraffin-embedded tumor tissues using two primary techniques. First, next-generation sequencing (NGS) is performed with a customized 37-gene panel to identify mutations in driver genes and variants of uncertain significance. Second, multiplex immunofluorescence (mIF) is utilized to target markers including HLA-DR, CD68, CD163, CD206, PD-L1, and PanCK. This enables the quantification of spatial distribution and density for specific immune cell subpopulations across various tumor regions. This integrated approach enables the simultaneous assessment of genomic heterogeneity and tumor-infiltrating immune cell characteristics. The resulting data identifies specific combinations of mutational profiles-such as EGFR status-and immune cell densities. These integrated combinations enable the study of their collective impact on patient survival, offering a promising approach for the development of future lung cancer prognostic models. This protocol demonstrates a robust method for characterizing the complex biological features of the LUAD tumor microenvironment.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844884","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment and Evaluation of a Sheep Model of Full-thickness Osteochondral Defect.","authors":"Raphaella Josino, José Ademar Villanova Junior, Fabiano Kupczik, Luiz Guilherme Achcar Capriglione, Bruna Schaidt, Fabiane Barchiki, Lidiane Maria Boldrini-Leite, Paulo Roberto Slud Brofman, Alejandro Correa, Marco Augusto Stimamiglio, Carmen Lúcia Kuniyoshi Rebelatto","doi":"10.3791/68842","DOIUrl":"https://doi.org/10.3791/68842","url":null,"abstract":"<p><p>Aging, trauma, genetic predisposition, and lifestyle impact the human body´s cartilage degradation. Once injured, continuous insults resulting from daily activities can trigger pathological conditions such as osteoarthritis, the primary cause of disability and socioeconomic loss worldwide. The main goal of all orthopedic surgeons treating joint cartilage injuries has been to reduce the extent of the lesion anatomically, repair the cartilage surface, and reestablish joint stability. Animal models are essential for the development of therapeutic drugs, but current models for cartilage defects are unsatisfactory. Osteochondral lesions in sheep are a valuable model for testing new therapies and biomaterials that can aid in the recovery of cartilage and bone in human joint environments. This study established an efficient protocol for inducing acute osteochondral defects in large animals. A standardized lesion was created in both medial femoral condyles. One knee was randomly assigned to receive gelatin-methacryloyl treatment, while the contralateral knee served as a control. Six months after the surgical procedure, the femoral condyle area was removed, the dissected knee joints were decalcified, embedded in paraffin, and cut into sections, which were stained with hematoxylin and eosin. Scores were used to evaluate the lesion. This methodology allows immediate macroscopic observation after injury induction. Additionally, this model effectively replicates clinical cartilage defects, providing a valuable model for studying their pathology and developing innovative therapeutic approaches.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844864","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Closed-Loop ta-VNS System Synchronized with BCI-Based Motor Training for Post-Stroke Upper Limb Rehabilitation.","authors":"Meilian Zhong, Yongchun Jiang, Siqi Huang, Peizhen Chen, Huijin Liu, Yongxiang Zhang, Xiaofei He, Fei Yang, Qiao Fu, Yuxin Zheng, Yuanyuan Guo, Qiang Lin","doi":"10.3791/69272","DOIUrl":"https://doi.org/10.3791/69272","url":null,"abstract":"<p><p>Transcutaneous auricular vagus nerve stimulation (ta-VNS) involves applying electrical stimulation via electrodes to the auricular concha. This activates vagal afferent fibers, initiating an ascending pathway from the periphery to the brainstem, which ultimately stimulates central vagal projections and promotes neural plasticity. Previous studies have demonstrated that combining ta-VNS with motor training offers synergistic benefits for motor recovery after stroke. However, these combined approaches typically employ open-loop stimulation with fixed parameters, lacking real-time closed-loop responsiveness to dynamic neural activity. To address this limitation, we developed a novel closed-loop ta-VNS system synchronized with electroencephalography (EEG)-triggered brain-computer interface (BCI) motor training. This system was designed to enhance corticospinal coupling and promote synaptic plasticity. We established a standardized protocol for applying this closed-loop ta-VNS system synchronized with BCI-based motor training in stroke patients. Using EEG-based functional assessment, we compared the effects of the closed-loop ta-VNS system synchronized with BCI-based motor training to those of sham ta-VNS synchronized with BCI-based motor training. This work provides the methodological and theoretical groundwork for the clinical application of this approach in stroke rehabilitation.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 230","pages":""},"PeriodicalIF":1.2,"publicationDate":"2026-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147844795","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}