Jove-Journal of Visualized Experiments最新文献

Erratum: Bulk Electroacupuncture Operation for Mice or Young Rats.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-18 DOI: 10.3791/6632

Siqi Yao, Yun Wei, Yuping Zhang, Xiaolin Yang, Zhifeng Wu

引用次数: 0

Erratum: Periorbital Placement of a Laser Doppler Probe for Cerebral Blood Flow Monitoring Prior to Middle Cerebral Artery Occlusion in Rodent Models.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-18 DOI: 10.3791/6629

Denali C Dickson, Mitchell J Bartlett, Sharon Hom, Helena W Morrison

引用次数: 0

Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using The Single-cell Microliter-droplet Culture Omics System.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-14 DOI: 10.3791/67925

Wenhsuan Chou, Xiaojie Guo, Liyan Wang, Xin-Hui Xing, Yi Wang, Chong Zhang

{"title":"Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using The Single-cell Microliter-droplet Culture Omics System.","authors":"Wenhsuan Chou, Xiaojie Guo, Liyan Wang, Xin-Hui Xing, Yi Wang, Chong Zhang","doi":"10.3791/67925","DOIUrl":"https://doi.org/10.3791/67925","url":null,"abstract":"Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors are hindered by cumbersome procedures and low throughput, impeding the rapid progress of microbial culturomics research. To address these challenges, we had successfully developed the Single-cell Microliter-droplet Culture Omics System (MISS cell), an automated high-throughput platform that utilizes droplet microfluidic technology for microbial monoclonal isolation, cultivation, and screening. This system can generate a large number of single-cell droplets and cultivate, screen, and collect monoclonal colonies in a short time, facilitating an integrated process from microbial isolation to picking. In this protocol, we demonstrated its application using the isolation and cultivation of human gut microbiota as an example and compared the microbial isolation efficiency, monoclonal culture performance, and screening throughput using the solid-plate culture method. The experimental workflow was simple, and reagent consumption was very low. Compared to solid-plate culture methods, the MISS cell could cultivate a greater diversity of gut microbiota species, offering significant potential and value for microbial culturomics research.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143755310","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}

引用次数: 0

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-14 DOI: 10.3791/67657

Zelvera Ismetova Usheva, David Leander Petersen, Torben Barington, Peter Mouritzen, Mike Bogetofte Barnkob

{"title":"Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level.","authors":"Zelvera Ismetova Usheva, David Leander Petersen, Torben Barington, Peter Mouritzen, Mike Bogetofte Barnkob","doi":"10.3791/67657","DOIUrl":"https://doi.org/10.3791/67657","url":null,"abstract":"The assessment of the cytotoxic potential of T cell-based therapies, such as chimeric antigen receptor (CAR) T cell treatments, is instrumental in assessing their efficacy and is a prerequisite for clinical application. However, traditional cytotoxicity assays are conducted as bulk assays and do not provide detailed information about the functional heterogeneity of the CAR T cell population. In this study, we describe a double-emulsion droplet-based method that allows for large-scale co-encapsulation of single effector CAR T cells with single target cells while enabling dual quantification of both cytotoxic effector molecules from T cells and cell death of the target cell. The protocol outlines a method for the generation and purification of CD19-specific CAR T cells, followed by their co-encapsulation in droplets with the CD19+ cell line JeKo-1, along with reagents to visualize cytotoxic effector molecule secretion (Granzyme B) and cell death (using propidium iodide, PI). We demonstrate how to generate droplets containing single CAR T and target cells using a commercially available microfluidics device for generating double emulsion droplets. Additionally, we provide examples of how to assay the functional diversity of CAR T cells in droplets using standard flow cytometry equipment. Finally, we briefly describe the temporal kinetics and heterogeneity of CD19-specific CAR T-cell killing. While this method focuses on cell death following a CAR T cell attack, it is also adaptable for examining other types of T cells, cytotoxic immune cells, and effector cell functions, such as cytokine secretion.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143755286","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}

引用次数: 0

Establishment of an Animal Training Model of Duodenoscopy, Choledochoscopy, and Laparoscopy for Cholelithiasis.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-14 DOI: 10.3791/67611

Baifeng Qian, Junlong Lin, Jianpeng Cai, Zhichao Li, Yunpeng Hua

{"title":"Establishment of an Animal Training Model of Duodenoscopy, Choledochoscopy, and Laparoscopy for Cholelithiasis.","authors":"Baifeng Qian, Junlong Lin, Jianpeng Cai, Zhichao Li, Yunpeng Hua","doi":"10.3791/67611","DOIUrl":"https://doi.org/10.3791/67611","url":null,"abstract":"Cholelithiasis is a condition characterized by the formation of stones in the gallbladder and/or bile ducts. Proficient use of duodenoscopy, laparoscopy, and choledochoscopy techniques plays a pivotal role in managing cholelithiasis. To enhance young doctors' proficiency in multiendoscope technology application, a training model for duodenoscopy, choledochoscopy, and laparoscopy was established using a live pig model. Initially, the duodenoscope was used to access the duodenum through passage through the mouth, esophagus, and stomach to observe the duodenal papilla. Subsequently, the tube was inserted into the common bile duct, followed by the placement of the guide wire. Trocars were subsequently inserted to establish pneumoperitoneum and facilitate the performance of laparoscopic cholecystectomy. The common bile duct was explored and opened with the retrieval of the guide wire found within it. The choledochoscope was then used to explore both distal common bile duct and intrahepatic bile ducts. Finally, under duodenoscopy guidance, either nasobiliary tube or plastic biliary stent placement occurred before primary suture closure of the common bile duct. Establishing a multi-scopy combined animal model can further enhance young doctors' proficiency in multi-scopy combined technology through practical experience.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143755292","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}

引用次数: 0

Production of Arbuscular Mycorrhizal (AM) Fungal Inoculum and Phenotypic Evaluation of Rice and AM Symbiosis Under Saline Conditions.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-14 DOI: 10.3791/67580

Wan-Ning Kuo, Pei-Jung Chen, Zheng-Lin Guo, Man-Chi Ho, Chao-Ling Ting, Shu-Yi Yang

{"title":"Production of Arbuscular Mycorrhizal (AM) Fungal Inoculum and Phenotypic Evaluation of Rice and AM Symbiosis Under Saline Conditions.","authors":"Wan-Ning Kuo, Pei-Jung Chen, Zheng-Lin Guo, Man-Chi Ho, Chao-Ling Ting, Shu-Yi Yang","doi":"10.3791/67580","DOIUrl":"https://doi.org/10.3791/67580","url":null,"abstract":"Rice (Oryza sativa L.) is a vital food crop for more than half of the global population. However, its growth is severely impacted by saline soils, which present a significant challenge to crop production worldwide. Arbuscular mycorrhizal (AM) fungi, which form mutualistic symbiotic relationships with over 90% of agricultural plants and 80% of terrestrial plant species, have been shown to enhance the salt tolerance of rice plants. AM fungi are obligate symbionts that cannot complete their life cycle without a host root. Therefore, effectively utilizing plants to produce AM fungal inoculum is crucial for advancing research in this field. In this study, we present a series of robust methods that begin with generating sand inoculum containing spores of Rhizophagus irregularis using Allium tuberosum L. These methods include inoculating rice seedlings with the sand inoculum, analyzing the growth phenotype of mycorrhizal rice, and quantifying fungal colonization levels using trypan blue staining under salt stress. These approaches can efficiently generate AM fungal inoculum for further investigation into how AM symbiosis enhances the salinity tolerance of rice.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143755397","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}

引用次数: 0

Effects of Mechanical Methods Used in Peri-implantitis Treatment on Implant Surface Decontamination and Roughness.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-14 DOI: 10.3791/67778

Ipek Ozgu, Kemal Ustun

{"title":"Effects of Mechanical Methods Used in Peri-implantitis Treatment on Implant Surface Decontamination and Roughness.","authors":"Ipek Ozgu, Kemal Ustun","doi":"10.3791/67778","DOIUrl":"https://doi.org/10.3791/67778","url":null,"abstract":"Various mechanical methods have been proposed for decontaminating dental implant surfaces with varying success. This in vitro study evaluated the decontamination efficiency of an air abrasion (AA) system with erythritol powder, a polyether-ether-ketone (PEEK) ultrasonic tip, and titanium curettes (TIT) and their effects on implant surface topography using scanning electron microscopy (SEM). A total of 60 implants were stained with permanent red ink and placed in 3D-printed Class 1A and Class 1B peri-implantitis defects, forming six groups (n=10 per group) based on defect type and treatment protocol. Additionally, one positive and one negative control implant was used. Erythritol powder, PEEK ultrasonic tips, and titanium curettes were applied for 2 min in Class 1A defects and 3 minutes in Class 1B defects. Residual red ink areas were quantified with digital software, and implant surface changes were analyzed using SEM and EDS. None of the methods achieved complete decontamination. However, erythritol powder was significantly the most effective, leaving a residual ink rate of 24% ± 6% (p < 0.001). PEEK ultrasonic tips resulted in 41% ± 4% residual ink, while titanium curettes left 55% ± 3%. Significant differences were observed among all methods. No significant difference in decontamination efficacy was found between Class 1A and Class 1B defects. SEM analysis showed minimal surface damage with erythritol powder and PEEK tips, whereas titanium curettes caused moderate to severe damage. Based on both decontamination efficiency and surface preservation, erythritol powder and PEEK tips are safe and effective options for peri-implantitis treatment, while titanium curettes are less effective and cause considerable surface damage. These findings may assist clinicians in peri-implantitis treatment planning.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143755291","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}

引用次数: 0

Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-14 DOI: 10.3791/67921

Olivia L Bossardet, Joseph M Holden, Lauren K Wareham

{"title":"Measuring Uptake of the Glucose Analog, 6-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-6-Deoxyglucose, in Intact Murine Neural Retina.","authors":"Olivia L Bossardet, Joseph M Holden, Lauren K Wareham","doi":"10.3791/67921","DOIUrl":"https://doi.org/10.3791/67921","url":null,"abstract":"The retina is a highly metabolic tissue with multiple cell types requiring glucose and its derivatives to produce energy in the form of ATP. Retinal cells, including endothelial cells, neurons, photoreceptors, and glial cells, express glucose transporters (GLUTs; e.g., GLUT1-4) to enable the uptake of glucose for energy production. GLUT1 is the most abundantly expressed glucose transporter in the retina. This protocol enables researchers to measure the uptake of glucose in the neural murine retina in ex vivo conditions using the fluorescent glucose analog 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose (6-NBDG). After retinal dissection, total retinal 6-NBDG levels can be easily determined via fluorescence endpoint measurement using a plate reader. For consistency, we recommend normalizing results to total protein levels. Although 6-NBDG is highly specific for GLUT1, uptake of this analog is detected in the presence of GLUT1 inhibitor BAY-876. As such, this assay provides a relatively quick and inexpensive method to measure glucose uptake ex vivo in whole mouse neural retina, which is partially mediated by GLUT1.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143755391","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}

引用次数: 0

Open-source Micro-Manager Plugin for Live-view Imaging of Fluorescent Dipoles.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-14 DOI: 10.3791/67755

Qin Ke, Suyi Zhong, Ying Chen, Dayong Jin, Peng Xi, Karl Zhanghao

{"title":"Open-source Micro-Manager Plugin for Live-view Imaging of Fluorescent Dipoles.","authors":"Qin Ke, Suyi Zhong, Ying Chen, Dayong Jin, Peng Xi, Karl Zhanghao","doi":"10.3791/67755","DOIUrl":"https://doi.org/10.3791/67755","url":null,"abstract":"Fluorescence polarization microscopy (FPM) can image the position and dipole orientation of fluorophores. Despite the achievements of super-resolution fluorescence polarization microscopy, their reliance on post-acquisition hinders real-time observation. Polarized structured illumination microscopy (pSIM) offers super-resolution imaging of fluorescent dipoles with fast imaging speed and is well-suited for live-cell applications. We developed an open-source implementation for real-time reconstruction of polarization images and display of the fluorescent dipoles. Additionally, we extended the method to achieve 3D orientation mapping (3DOM), broadening its utility for complex biological studies.Furthermore, we have presented a thorough introduction to extending an existing SIM microscope on polarization imaging and provided a detailed configuration guide of Micro-Manager 2.0 to control the microscope, enabling real-time preview of polarized imaging. Additionally, we have provided the MATLAB code for full reconstructionencompassing both pSIM and 3DOM. This comprehensive guide aims to assist beginners in quickly mastering and easily getting started with the operations.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143755395","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}

引用次数: 0

Direct Vagus Nerve Injection Protocol For Rats.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-03-14 DOI: 10.3791/67820

Nanda L Regmi, Xin Chen, Rachel M Bailey, Steven J Gray

{"title":"Direct Vagus Nerve Injection Protocol For Rats.","authors":"Nanda L Regmi, Xin Chen, Rachel M Bailey, Steven J Gray","doi":"10.3791/67820","DOIUrl":"https://doi.org/10.3791/67820","url":null,"abstract":"There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly to the peripheral nervous system is less common, with fewer detailed methods publications available to aid researchers. Here, we describe a direct nerve injection method for peripheral nervous system drug delivery, using the vagus nerve as a model nerve. This method can be used in the treatment of autonomic nervous system disorders through targeting of the left vagus nerve, although this general injection method can be extrapolated to injection of other nerves with minor modification. This method explains all critical steps involved in the procedure involving microsurgery in anesthetized adult rats under a dissecting microscope. The use of a tracking dye is described to facilitate the monitoring of injection fidelity in real time. Illustrations of successful and failed injections are provided. If carried out properly, direct vagus nerve injections can be conducted in a safe manner that is well-tolerated by the rat without post-delivery complications. For example, once the surgeons were trained in this method, six out of six rats were successfully injected without any complications. This method of direct nerve injection for preclinical rat studies is capable of delivering agents (including but not limited to gene therapy) to peripheral nerves.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143755313","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}

引用次数: 0