Jove-Journal of Visualized Experiments最新文献

{"title":"Lipid Exchange Assay in Living Cells.","authors":"Sanjna Rana, Antonio Torlentino, Pavana Suresh, W Todd Miller, Erwin London","doi":"10.3791/68008","DOIUrl":"https://doi.org/10.3791/68008","url":null,"abstract":"<p><p>Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane's propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804498","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":"Picrosirius Red Staining for Semiquantitative Histopathologic Evaluation of Collagen Deposition in Murine Models of Chronic Lung Allograft Rejection.","authors":"Birte Ohm, Julia Thomé, Thierry Christmann, Maike Lind, Yoshito Yamada, Andreas Spörlein, Laura Anna Schneider, Steffen U Eisenhardt, Wolfgang Jungraithmayr","doi":"10.3791/66395","DOIUrl":"https://doi.org/10.3791/66395","url":null,"abstract":"<p><p>Fibrosis is the pathophysiologic hallmark of chronic rejection after lung transplantation and the foremost hurdle to long-term recipient survival. Several murine lung transplantation models are available for the study of chronic rejection. However, they display heterogeneous results regarding fibrotic changes of the graft, and the histologic extent of fibrosis is mostly reported qualitatively. Therefore, a spatially resolved approach that allows for statistical analysis can aid in evaluating fibrosis in these models. This study presents Picrosirius Red staining for a semiquantitative evaluation of collagen organization in murine lung allografts and compares it to standard Hematoxylin and Eosin, Masson's Trichrome, and Herovici's stains. Staining was performed on sections from two different murine transplantation models based on minor and major histocompatibility complex (MHC) mismatches. The method was established for semiquantitative analysis of collagen organization in whole lung sections. Thus, it can serve as a tool for murine experimental models of fibrotic lung diseases.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804516","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":"Separation and Identification of Conventional Microplastics from Farmland Soils.","authors":"Siyang Ren, Martine Graf, Kai Wang, Jinrui Zhang, Hanyue Zhang, Xiuting Liu, Jingjing Li, Tong Zhu, Kaige Ren, Yingming Sun, Ruimin Qi, Benjamin I Collins, Li Xu, Xiaoxu Jiang, Jixiao Cui, Fan Ding, Changrong Yan, Xuejun Liu, Davey L Jones, David R Chadwick","doi":"10.3791/67064","DOIUrl":"https://doi.org/10.3791/67064","url":null,"abstract":"<p><p>Microplastics (MPs) pollution in the terrestrial environment has received increasing attention over the last decade, with increasing studies describing the numbers and types of MPs in different soil systems and their impacts on soil and crop health. However, different MPs extraction and analytical methods are used, limiting opportunities to compare results and generate reliable evidence for industry advice and policymakers. Here, we present a protocol that describes the methodology for sampling, separation, and chemical identification of conventional MPs from soil. The method is low-cost, and the materials are readily available. This enhances operational ease and may help with widespread adoption. The protocol provides detailed information on sample collection from the top 0-30 cm of soil using plastic-free utensils; simulation of different soil types through the use of various solid media (such as bentonite clay, silicon dioxide, and non-contaminated soil), with the addition of the same mass of polyethylene(PE)-MPs for subsequent quantification; density separation of plastic particles utilizing saturated sodium chloride (NaCl) solution and digestion of organic impurities in the supernatant using 4 M sodium hydroxide (NaOH) solution; quantification of particles using fluorescent microscopy after Nile Red staining; and polymer identification using micro Fourier-Transform Infrared Spectroscopy (μ-FTIR) or Laser-Direct Infrared (LDIR) spectroscopy. The MPs recovery rate ranged from 83% - 90% for the abovementioned media. This protocol presents an efficient method for soil MPs analysis that is optimized for feasibility, applicability, and cost-effectiveness. Moreover, the video accompanied can guide the process of analyzing the soil MPs step-by-step virtually. This study is dedicated to standardizing the methods for soil MPs analysis, enhancing the connectivity and comparability of measurements, and establishing a foundation for more standardized and scientific research.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804540","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":"Quantification of Drosophila melanogaster Grooming Behavior for Evaluation of Excessive Grooming Phenotypes.","authors":"Theodore Hatfield, Seth Johnson","doi":"10.3791/67708","DOIUrl":"https://doi.org/10.3791/67708","url":null,"abstract":"<p><p>Observable changes in stereotyped grooming are applied translationally in model organisms. These changes are representative of pathologies that elicit similar deviations in human behavior; for example, excessive grooming acts as a proxy for obsessive and compulsive behaviors present in conditions like Tourette Syndrome or obsessive-compulsive disorder. The grooming assay presented allows for the evaluation of abnormal self-grooming phenotypes in Drosophila melanogaster. Flies are recorded for a period of 10 min, and these recordings are observed and annotated blind for previously defined grooming behaviors. Quantitative measures of both grooming bout frequency and the time spent engaging in self-grooming can be obtained by manually annotating the footage. The assay is relatively inexpensive, requires few materials not already available in laboratory environments, and is easily adaptable to fit the specific needs of any given study aiming to observe grooming. Additionally, the low level of skill needed to perform the assay, as compared to computer science-heavy automated methods, makes the protocol well-suited for small labs and students. We discuss in detail the steps required to perform this assay and its present limitations.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804521","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":"Genome Editing in the Yellow Fever Mosquito Aedes aegypti using CRISPR-Cas9.","authors":"Iliano V Coutinho-Abreu, Fangying Chen, Hsing-Han Li, Noah H Rose, Omar S Akbari","doi":"10.3791/67732","DOIUrl":"https://doi.org/10.3791/67732","url":null,"abstract":"<p><p>The emergence of the clustered, regularly interspersed, short palindromic repeats (CRISPR)-Cas9 technology has revolutionized the genetic engineering field and opened the doors for precise genome editing in multiple species, including non-model organisms. In the mosquito Aedes aegypti, loss-of-function mutations and DNA insertions have been accomplished with this technology. Here, we describe a detailed protocol for genome editing through embryonic microinjection in the mosquito A. aegypti using the CRISPR-Cas9 technology, focusing on both the generation of gene knockout and knockin lines. In this protocol, quartz needles are filled with a mixture of guide RNA, recombinant Cas9, and a plasmid containing a DNA cassette encoding a gene for a fluorescent marker, if gene knockin is desired. Embryos at the preblastoderm stage are lined up onto a strip of double-sided sticky tape placed onto a coverslip, which is subsequently mounted onto a glass slide. With the help of a microinjector, the needles are inserted gently into the posterior end of the embryos and a small volume of the CRISPR mixture is dispensed. When the embryos are hatched, the larvae are checked under the fluorescent scope, and the pupae are sex-sorted and separated in different cages. Once the adults emerge, these are reciprocally crossed with wild-type individuals, blood-fed, and placed for egg laying. Once these eggs are hatched, the fluorescent larvae collected represent individuals with stable insertion of the DNA cassette into their genome. These larvae are then grown to the adult stage, outcrossed to wild-type individuals, and then further assessed through molecular techniques to confirm that the exact sequence of the DNA cassette is present at the desired site of the mosquito genome. Homozygous lines can also be obtained by following the provided pipeline of crossing schema and molecular screening of the mutations.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804448","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":"Imaging Ca2+ Signals in Small Pulmonary Veins at Physiological Intraluminal Pressures.","authors":"Yen-Lin Chen, Kyosuke Kazama, Vihaan Vattipally, Swapnil K Sonkusare","doi":"10.3791/67722","DOIUrl":"https://doi.org/10.3791/67722","url":null,"abstract":"<p><p>Pulmonary veins (PVs) carry oxygen-rich blood from the lungs back to the left heart, thus serving an important function in the delivery of oxygen-rich blood to vital organs. However, most studies of pulmonary vasculature have focused on pulmonary arteries and capillaries under normal and disease conditions. Ca²⁺ signals are critical regulators of vascular function. Despite the critical physiological roles of PVs, Ca²⁺ signals in small intrapulmonary PVs have not been recorded under physiological conditions. Here, we describe a technique to record Ca²⁺ signal activity in mouse PVs isolated, cannulated and pressurized at 5 mmHg. By incorporating a Ca²⁺ indicator, we can study Ca²⁺ signals in the myocyte layer of small PVs using high-speed, spinning disk confocal imaging under physiological conditions. Our representative data indicates that the Ca²⁺ signals in small PV myocytes are mediated by openings of ryanodine receptor ion channels. This method will be of considerable interest to researchers in the field of pulmonary vascular physiology and disorders.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804453","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":"DiI Dye-Filling as a Simple and Inexpensive Tool to Visualize Ciliated Sensory Neurons in C. elegans.","authors":"Nicole M Andrews, Lexi Gerten, Angela Edwards, Maureen Brennan, Quinn Reynolds, Skye Bernstein, Tina Springer, Melissa LaBonty","doi":"10.3791/64052","DOIUrl":"https://doi.org/10.3791/64052","url":null,"abstract":"<p><p>C. elegans have long been used as a simple and accessible model to study neuronal structure and the many functions of the nervous system. Of the 302 neurons within the adult hermaphrodite nervous system, 60 are classified as ciliated sensory neurons. These neurons are central to a number of C. elegans behaviors, including but not limited to chemo-, mechano-, and osmosensing, male mating, and dauer formation. For several decades now, members of the C. elegans community have used the red fluorescent lipophilic dye DiI to visualize a subset of the ciliated sensory neurons that are directly exposed to the external environment. This dye enters the ciliated ends of the neurons and distributes in a relatively uniform pattern throughout the dendrites, cell bodies, and axons. This simple and powerful method makes an excellent first-pass tool to identify genetic mutants that impart structural or functional defects in ciliated sensory neurons. Here, we present a streamlined version of this staining method to visualize the eight pairs of amphid and two pairs of phasmid neurons that are environmentally exposed in C. elegans. We discuss tips for using this inexpensive method for imaging cellular dye-filling patterns in anesthetized animals.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804221","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":"Techniques for Lower Trapezius Transfer Using Achilles Allograft in Irreparable Posterosuperior Rotator Cuff Tears.","authors":"Chen-Heng Hsu, Chieh-An Chuang, Huan Sheu, You-Hung Cheng, Cheng-Pang Yang, Yi-Hsuan Lin, Joe Chih-Hao Chiu","doi":"10.3791/67899","DOIUrl":"https://doi.org/10.3791/67899","url":null,"abstract":"<p><p>The management of irreparable rotator cuff tears presents significant challenges, particularly in active individuals experiencing functional limitations, such as reduced forward elevation and deficits in both external and internal rotation. Traditional latissimus dorsi (LD) tendon transfer has shown effectiveness in reducing pain associated with posterosuperior cuff tears but often yields inconsistent functional outcomes. This is largely due to the LD's primary role as an internal rotator, which limits its capacity to restore normal shoulder biomechanics. To address these limitations, the lower trapezius (LT) tendon transfer, augmented with an Achilles allograft, has emerged as an alternative to enhance external rotation, leveraging the LT's line of pull, which closely resembles that of the infraspinatus muscle. This protocol outlines a modified surgical technique for LT tendon transfer with Achilles allograft augmentation, detailing patient positioning, tendon harvest, graft preparation, arthroscopic passage, and fixation methods. The protocol emphasizes key anatomical landmarks to minimize neurovascular injury and enhance graft integration. Postoperative care includes a 3 month immobilization period followed by a structured rehabilitation program to facilitate functional recovery. This procedure is indicated for a specific patient group requiring improved external rotation and is biomechanically advantageous over the LD transfer. Though additional studies are warranted to confirm its efficacy in broader patient populations, early clinical outcomes suggest that LT transfer with Achilles allograft could offer superior biomechanical alignment and improved external rotation.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804542","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":"Block Building Task Identifies Distinct Groups of Left/Right-hand Choice Patterns After Unilateral Peripheral Nerve Injury.","authors":"Taewon Kim, Summer Fletcher, Claudia Gonzalez, Benjamin A Philip","doi":"10.3791/66919","DOIUrl":"https://doi.org/10.3791/66919","url":null,"abstract":"<p><p>Numerous methods exist to assess hand and arm function after upper extremity peripheral nerve injury, but peripheral injuries are often unilateral, and few existing methods are designed to capture the unique consequences of unilateral injury. Unilateral impairment of an upper extremity can lead to increased or decreased use of the dominant hand, and either change may be adaptive or maladaptive depending on the individual patient's needs. To identify atypical hand usage (left/right choices), researchers and clinicians need to measure it. However, hand usage is traditionally assessed with self-report surveys, which do not necessarily reflect actual left/right-hand choices. Here, this gap in knowledge is addressed with the Block Building Task (BBT), which provides a rapid, quantitative, inexpensive assessment of left/right-hand choices in an unconstrained environment. In the BBT, participants build abstract shapes with interlocking plastic bricks, with no instructions about hand usage. The primary outcome is the fraction of reaches (i.e., for the initial pickup of each brick) made with each hand. After unilateral peripheral nerve injury, patients fell into three clusters: approximately typical hand use (44%), always use the dominant hand (44%), or never use the dominant hand (13%). Even among patients with an injured dominant hand, atypically elevated use of the dominant hand occurred regularly (36%). Notably, hand usage was not predicted by clinical characteristics, so the BBT provides an objective measurement of left/right-hand choices that are not otherwise predictable from the clinical characteristics of patients with peripheral nerve injury. The BBT protocol will be of interest to researchers or clinicians interested in the assessment of conditions with asymmetric effects on the upper limb.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804210","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":"In Vitro Thrombosis Test for Ventricular Assist Devices.","authors":"Mansur Zhussupbekov, Scott Stelick, Rugveda Thanneeru, Shivbaskar Rajesh, Salim E Olia, Harvey S Borovetz, James F Antaki","doi":"10.3791/67731","DOIUrl":"https://doi.org/10.3791/67731","url":null,"abstract":"<p><p>The risk of thrombosis remains a significant concern in the development and clinical use of ventricular assist devices (VADs). Traditional assessments of VAD thrombogenicity, primarily through animal studies, are costly and time-consuming, raise ethical concerns, and ultimately may not accurately reflect human outcomes. To address these limitations, we developed an aggressive in vitro testing protocol designed to provoke thrombosis and identify potential high-risk areas within the blood flow path. This protocol, motivated by the work of Maruyama et al., employs a modified anticoagulation strategy and utilizes readily available components, making it accessible to most laboratories conducting in vitro blood testing of VADs. We demonstrated the utility of this method through iterative testing and refinement of a miniature magnetically levitated pediatric VAD (PediaFlow PF5). The method has been effective in identifying thrombogenic hotspots caused by design and manufacturing flaws in early VAD prototypes, enabling targeted improvements before advancing to animal studies. Despite its limitations, including the absence of pulsatile flow and the influence of donor blood characteristics, this protocol serves as a practical tool for early-stage VAD development and risk mitigation.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 217","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143804489","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}