Jove-Journal of Visualized Experiments最新文献

{"title":"Comprehensive Analysis of Procoagulant Platelets Exhibiting Features of Necrosis, Apoptosis and Platelet Activation.","authors":"Paresh P Kulkarni, Keith R McCrae","doi":"10.3791/68116","DOIUrl":"https://doi.org/10.3791/68116","url":null,"abstract":"<p><p>Platelets circulating in the bloodstream are relatively quiescent but become \"activated\" upon encountering stimulants or \"agonists\" at the site of blood vessel injury. Proaggregatory and procoagulant platelets represent two distinct populations of activated platelets. While proaggregatory platelets facilitate the cessation of bleeding, or \"hemostasis,\" by forming a plug of platelets clumped together through fibrinogen bridges, procoagulant platelets dramatically accelerate the coagulation cascade, culminating in fibrin clot formation. An interesting aspect of procoagulant platelets is that their morphology exhibits certain features of \"necrosis\" and \"apoptosis.\" They may thus represent a form of cell death in platelets, albeit with an important role in thrombosis and hemostasis. This article introduces the concept of procoagulant platelets, their relevance to health and disease, and a comparison of existing methods for their analysis. It then provides comprehensive protocols for analyzing procoagulant platelets, investigating the mechanisms of their formation, and assessing their prothrombotic role in facilitating coagulation. The article concludes with a discussion of key steps, limitations, and troubleshooting principles for the described methods.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259169","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":"Structural Biology and Analytical Chemistry Approaches for Characterizing C-Glycoside Metabolic Enzymes in Human Gut Microbiota.","authors":"Pengfei He, Yingxin Shao, Shuqi Zhou, Rufeng Wang, Wenfu Ma","doi":"10.3791/67629","DOIUrl":"https://doi.org/10.3791/67629","url":null,"abstract":"<p><p>The gut microbiota has emerged as a key modulator of host health, particularly because of its capacity to metabolize dietary glycosides. While C-glycosides demonstrate superior chemical stability and resistance to enzymatic cleavage compared to O-glycosides, specialized gut bacteria can selectively metabolize them, improving their bioavailability. Since the identification of microbial C-glycosidase enzymes, the precise reaction mechanisms remain incompletely understood, and standardized methodologies for studying these processes are still not well-established. To address this gap, we introduced an integrated multidisciplinary approach combining structural biology, enzymology, and analytical techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR), to systematically characterize these enzymatic activities. This comprehensive approach enables precise characterization of C-glycoside metabolism at molecular, enzymatic, and metabolic levels.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259193","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":"Cell-Free Dot Blot as a Practical and Adaptable Immunoassay Platform for the Detection of Antibody Response in Human and Animal Sera.","authors":"Masoud Norouzi, Riham Zayeni, Serena Singh, Keith Pardee","doi":"10.3791/67973","DOIUrl":"https://doi.org/10.3791/67973","url":null,"abstract":"<p><p>The string of global pathogenic outbreaks over the past two decades has highlighted the importance of serosurveillance strategies. Immunoassay platforms that serve to detect disease-specific antibodies in patients' sera are at the core of serosurveillance. Common examples include enzyme-linked immunosorbent assays and lateral flow assays; however, while these are gold standard methods, they require pathogen-specific consumables and specialized equipment, which limits their use outside of well-resourced laboratories. We have recently developed a novel immunoassay platform called Cell-Free Dot-Blot (CFDB) and validated it using human and animal sera against SARS-CoV-2. Unlike conventional immunoassays, CFDB patient serum samples are immobilized to a solid phase (nitrocellulose membrane), while the target antigen is suspended in the mobile phase of the assay. To improve access to serosurveillance capabilities, CFDB antigens are produced on demand and with low-burden infrastructure using in vitro protein expression. Here, the antigen is fused with a peptide tag that can be detected using a single universal reporter protein for any CFDB assay. The result is that the CFDB does not require access to a multi-well plate reader or purified commercial molecular assay components. With these design considerations, CFDB addresses the shortcomings of existing immunoassay platforms by providing accessibility to non-centralized laboratories, adaptability for emerging pathogens, and affordability for lower-income communities. In the current article, we will provide a step-by-step protocol to prepare and perform a CFDB immunoassay. Using our recent work on SARS-CoV-2 CFDB as an example, we will cover antigen DNA design for on-demand cell-free production, followed by preparation of the CFDB reporter protein, immobilization of serum samples on the solid phase, and finally, antigen-binding and detection steps of the assay. We anticipate that by following these instructions, researchers will be able to adapt the CFDB assay to detect immune responses in human and animal sera to any given pathogen.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259168","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":"Identification of Pre-weanling 7-Day-Old Mice with Pinna Edge Biopsies.","authors":"Diane Chen, Denise Molk, Joanne Morris, Corinna Beale","doi":"10.3791/68299","DOIUrl":"https://doi.org/10.3791/68299","url":null,"abstract":"<p><p>Identification and genotyping of mice is often an essential part of many in vivo scientific studies. Several methods have been described for identification and genotyping in weaned mice; however, to date, there are far fewer techniques described for pre-weanling mice. Pinna Edge Biopsy (PEB) is a method of identification with subsequent use of tissue for genotyping. This article describes and provides video instruction on a stepwise approach to PEB on post-natal day (PND) 7 mice. Eight different patterns of pinna biopsy location and technique are outlined which have been shown to sustain into adulthood for identification. This article also discusses ways to optimize the PEB and shows the benign effect this technique has on the growth and development of the pups, with all mice showing appropriate weight gain without any morbidity or mortality throughout the study. This technique will provide investigators with a method for mouse identification at an age where there are few identification and genotyping methods available.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259175","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":"High-Resolution C. elegans Imaging Across All Larval Stages.","authors":"Simon Berger, Silvan Spiri, Andrew deMello, Alex Hajnal","doi":"10.3791/68172","DOIUrl":"https://doi.org/10.3791/68172","url":null,"abstract":"<p><p>Caenorhabditis elegans has become one of the most widely studied and best-understood animal models in biology. Three features are key to C. elegans' success as a model organism: its invariant cell lineage, transparency, and genetic tractability. These render it ideal for a diverse range of microscopy-based studies directly in vivo. Live C. elegans larvae and adults often need to be immobilized during image acquisition. Traditional immobilization methods adversely affect animal development, especially in time-lapse imaging applications. Here, a detailed setup and operation protocol for a novel microfluidic imaging method is introduced, which addresses the limitations associated with traditional agar-pad-based immobilization and other microfluidic strategies. This approach enables simultaneous live imaging across various larval stages while preserving worm orientation and identity over time. To achieve this, a microfluidic trap channel array is employed, with its geometry precisely designed to maintain a stable worm orientation while accommodating growth and molting. Immobilization is facilitated by an active hydraulic valve that applies pressure to secure worms against the cover glass solely during image acquisition. This design allows high-resolution imaging with minimal effects on worm viability or developmental timing.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259174","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":"Novel Triple-Loop Technique for Suturing TFCC Injuries without Transosseous Tunnel.","authors":"Chao Jiang, Xin Li, SongOu Zhang, YuKe Chen, Ping Zhou, XuJun Hu","doi":"10.3791/68077","DOIUrl":"https://doi.org/10.3791/68077","url":null,"abstract":"<p><p>Injuries to the triangular fibrocartilage complex (TFCC) are the most common cause of ulnar-sided wrist pain. As deepening of the research on the function and role of the TFCC, various treatment methods have emerged. Surgical treatment is often required for TFCC injuries that do not respond to conservative therapy. Currently, suturing techniques for TFCC injuries primarily include intra-capsular and transosseous tunnel suture techniques. However, traditional intra-capsular suturing has limited effectiveness for deep tears with a foveal footprint contact, while transosseous tunnel techniques pose problems such as difficulty in locating tunnels and ulna fracture. We have developed an improved triple-loop technique for treating Palmer IB-type TFCC injuries. This technique allows for suturing of both the deep and superficial layers of the TFCC without the need for a transosseous tunnel. Clinical follow-up results show that this is a safe, effective, and simple surgical method for treating TFCC injuries. In this article, we also discussed the comparison between the triple-loop surgical approach and other commonly used surgical approaches.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259183","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":"Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells.","authors":"Seth W Croslow, Timothy J Trinklein, Siheun Lee, Stanislav S Rubakhin, Jonathan V Sweedler","doi":"10.3791/68376","DOIUrl":"https://doi.org/10.3791/68376","url":null,"abstract":"<p><p>Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry (MS) is capable of label-free, high-throughput characterization of endogenous molecules in individual cells. The recent advances in the development of MALDI mass spectrometers with laser-induced post-ionization (MALDI-2) provide greatly enhanced sensitivity of detection for a variety of lipids and other small molecules. However, MS imaging of large samples with MALDI-2 at cellular resolution is prohibitively slow for most applications. In this protocol, primary cells are isolated and dispersed onto conductive slides. Relative cell locations are determined by whole-slide fluorescence microscopy, followed by accurate coregistration of the microscopy coordinates to the stage coordinates of the MALDI-2 mass spectrometer. Targeted MS analysis of only cell locations provides high-throughput, single-cell measurements with high analyte coverage and reduced data size as compared to MS imaging of the entire sample. We describe the critical steps necessary for single-cell preparation, whole-slide fluorescence imaging, matrix application, and MALDI-2 mass spectrometry.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259172","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 Location Approach with Silk Thread Guided Laparoscopic Segmentectomy for Liver Tumor.","authors":"Yifan Zhou, Zhihong Zhang, Haotian Liao, Yong Zeng, Jiaxin Li","doi":"10.3791/68395","DOIUrl":"https://doi.org/10.3791/68395","url":null,"abstract":"<p><p>When performing hepatectomy to treat liver tumors, accurately determining the resection margin and ensuring the adequacy of residual liver parenchyma are of utmost importance. At present, intraoperative ultrasound and indocyanine green fluorescence navigation are frequently utilized methods. However, certain technical constraints prevent their extensive application. We have developed the 3D-LAST technique for precise liver tumor resection. This technique makes use of computer post-processing to extract features from computed tomography (CT) scans and generate volumetric images, creating three-dimensional (3D) visualizations. This provides a valuable resource for clinical decision-making, as it can vividly display complex internal anatomical structures in an intuitive and stereoscopic manner. In this study, preoperative 3D positioning was carried out on patients with a single liver tumor to identify anatomical landmarks and calculate the resection range. During the surgical procedure, margin lines of lengths calculated by preoperative 3D software were set up, and silk thread was used to mark the edges. This approach offers a time-saving and accurate way to determine the optimal cutting plane. The objective of this article is to demonstrate the viability of applying 3D-LAST in laparoscopic segmentectomy for liver tumors. The results of the research indicate that 3D-LAST is a safe, effective, and practical new method for intraoperative liver navigation, and it has great potential for widescale promotion.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259195","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":"Laparoscopic-Assisted Seldinger Technique for Peritoneal Dialysis Catheter Insertion.","authors":"Cheng-Yen Chen, Ming-Tsun Tsai, Niang-Cheng Lin, Chih-Ching Lin, Szu-Yuan Li","doi":"10.3791/67988","DOIUrl":"https://doi.org/10.3791/67988","url":null,"abstract":"<p><p>The management of kidney failure has advanced considerably, with peritoneal dialysis (PD) established as a reliable treatment option that uses the peritoneal membrane to remove excess fluid and toxins. PD has shown particular benefits in the initial stages of treatment, potentially leading to better outcomes compared to hemodialysis. Successful PD, however, depends heavily on the correct placement of the PD catheter. This study presents a protocol to perform the laparoscopic-assisted Seldinger technique (LAST) for peritoneal dialysis catheter insertion, highlighting a combination of the Seldinger method's minimal invasiveness with the enhanced visualization of laparoscopy. LAST allows precise catheter placement, making it especially suitable for patients with prior abdominal surgery or those requiring urgent-start PD. This technique enables patients to initiate PD within a day of catheter insertion. The post-surgical protocol involves a gradual increase in dialysate volume, allowing patients to achieve a full PD dose by day five while maintaining controlled peritoneal pressure and minimizing the risk of dialysate leaks. By integrating laparoscopic visualization with the minimally invasive Seldinger technique, LAST has the potential to offer a superior alternative to traditional methods.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259177","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":"Live-Cell Förster Resonance Energy Transfer Imaging of Metabolically Regulated Akt Activation Dynamics in HepG2 Cells.","authors":"Javed Akhtar, Muhammad Imran, Jiahe Wang, Gang Ma, Guanyu Wang","doi":"10.3791/68075","DOIUrl":"https://doi.org/10.3791/68075","url":null,"abstract":"<p><p>Metabolically regulated Akt activation is a critical node in the insulin signaling cascade and provides valuable insights into the relationship between diabetes and cancer. To precisely quantify Akt activity in HepG2 cells, we developed a robust, reproducible protocol utilizing Förster Resonance Energy Transfer (FRET) with genetically encoded Akt-specific biosensors. This protocol outlines detailed steps for cell culture, imaging dish preparation, and transfection of HepG2 cells to express FRET-based biosensors, alongside specific guidelines for laser scanning confocal microscope hardware and software configuration. The results demonstrated unique patterns of insulin signaling in HepG2 cells, which exhibit an irreversible switch characterized by constitutive Akt activation with a defined switch-on threshold but no switch-off threshold. In contrast, myotubes display a reversible switch. The persistent Akt activation in HepG2 cells suggests mechanisms underlying insulin resistance and metabolic dysregulation in hepatic cells, with broader implications for understanding the progression of metabolic disorders and cancer. This protocol offers a valuable framework for exploring Akt-related signaling pathways and cellular behaviors across various disease contexts.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259178","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}