Jove-Journal of Visualized Experiments最新文献

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67479

Yannick Masson, Aude Sivery, Corentin Spriet, Anthony Treizebre, Christophe Biot, Cedric Lion

{"title":"Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy.","authors":"Yannick Masson, Aude Sivery, Corentin Spriet, Anthony Treizebre, Christophe Biot, Cedric Lion","doi":"10.3791/67479","DOIUrl":"https://doi.org/10.3791/67479","url":null,"abstract":"Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections. Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques. In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494292","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

The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67805

Anjali S Yennemadi, Joseph Keane, Gina Leisching

{"title":"The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood.","authors":"Anjali S Yennemadi, Joseph Keane, Gina Leisching","doi":"10.3791/67805","DOIUrl":"https://doi.org/10.3791/67805","url":null,"abstract":"Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single population, as has been done historically. In particular, low-density and normal-density neutrophils (LDNs, NDNs) have been shown to have functionally and metabolically distinct profiles, a factor that must be considered when publishing neutrophil research. Here, we present a modified method for the untouched isolation and separation of LDNs and NDNs from whole blood. The density gradient medium (1.135 g/mL) is combined at 9:10 with 10x PBS. Specific density gradients of 55%, 70%, and 81% are subsequently made by combining the 100% density gradient medium with 1x phosphate-buffered saline (PBS). Neutrophils isolated from 12 mL of peripheral whole blood obtained from consented donors using a negative selection-based magnetic isolation kit are resuspended in the 55% fraction. A volume of 3 mL of the 81% and 70% fractions is layered into a 15 mL tube, followed by the 55% fraction containing total neutrophils. The density gradients are then centrifuged at 720 x g for 30 min. Two distinct bands are obtained at the 55%/70% interface (LDNs) and 70%/81% interface (NDNs). The cells are carefully pipetted into separate tubes and washed using PBS. The purity of the isolated fractions is determined using flow cytometry. Both LDNs and NDNs were defined as CD14lo CD15+ SSChi by flow cytometry. Isolation purity was calculated at ≥93% of viable cells for both types. This method provides a reliable and efficient approach for separating LDN and NDNs from peripheral blood, ensuring high purity and viability of the isolated cells. Enhancing the precision of neutrophil isolation facilitates more accurate downstream analyses of these distinct neutrophil subpopulations. These are critical for advancing our understanding of neutrophil heterogeneity and its implications in various physiological and pathological contexts.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143493749","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

Use of In Vivo Assembly for High-efficiency Plasmid Construction.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/67870

Hannah G Braun, Jenny-Lee Thomassin

引用次数: 0

The Mouse Stroke Unit Protocol with Standardized Neurological Scoring for Translational Mouse Stroke Studies.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-02-07 DOI: 10.3791/66847

Michail Georgopoulos, Angelos Pavlopoulos, Nefeli Zerva, Asterios Kokkonakis, Iordanis Mourouzis, Nikolaus Plesnila, Constantinos Pantos, Athanasios Lourbopoulos

{"title":"The Mouse Stroke Unit Protocol with Standardized Neurological Scoring for Translational Mouse Stroke Studies.","authors":"Michail Georgopoulos, Angelos Pavlopoulos, Nefeli Zerva, Asterios Kokkonakis, Iordanis Mourouzis, Nikolaus Plesnila, Constantinos Pantos, Athanasios Lourbopoulos","doi":"10.3791/66847","DOIUrl":"https://doi.org/10.3791/66847","url":null,"abstract":"The filament model of middle artery occlusion (fMCAo) is perhaps the most translational mouse stroke model, allowing for controlled ischemia with intravascular reperfusion/recanalization. However, it lacks alignment with current clinical advances for stroke care (e.g., Stroke Units), usually employs subjective or vague neurological scoring among laboratories, and exhibits high acute-phase mortality. Here, we address these limitations with validated video-guided protocols. We present the mouse Stroke Unit (mSU) protocol with instructional videos and a decision algorithm (Risk Stratification Score), bridging the gap between clinical and mouse stroke modeling. To increase accuracy and sensitivity of stroke neurological scoring, we present for the first time a video-standardized format of the focal Experimental Stroke Scale (fESS) and prove its value up to 6 months post-stroke. Additionally, protocols for mice Ladder-rung test, as well as the known Cylinder test, for unbiased, quantitative assessment of limbs´ motor function are presented. Results highlight mSU's translational efficacy. Focal ESS (fESS) excels over other known scales in detecting focal stroke deficits, capturing recovery, and maintaining sensitivity for up to 6 months post-stroke. Ladder-rung and Cylinder tests objectively quantify and monitor fore- and hind-limb motor deficits, long-term. In summary, integrating mSU, fESS, and motor function tests provides a robust framework for clinically relevant stroke investigations. Our protocols improve the translational value in mouse stroke research.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143493677","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

Spinal Sonography for Ultrasound-Guided Lumbar Neuraxial Anesthesia.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-31 DOI: 10.3791/66901

Antonio Gonzalez Fiol, F N U Pawan, Aymen Alian

{"title":"Spinal Sonography for Ultrasound-Guided Lumbar Neuraxial Anesthesia.","authors":"Antonio Gonzalez Fiol, F N U Pawan, Aymen Alian","doi":"10.3791/66901","DOIUrl":"https://doi.org/10.3791/66901","url":null,"abstract":"Neuraxial anesthesia is one of the few remaining forms of regional anesthesia that relies on palpation and tactile feedback techniques to facilitate catheterization into the epidural space. Over two decades ago, spine ultrasonography was demonstrated to provide reliable guidance for locating the epidural space. Compared to the palpation technique, preprocedural ultrasonography has been shown to result in fewer needle punctures and fewer traumatic procedures, particularly in patients with abnormal or distorted spine anatomy (e.g., scoliosis, obesity). Despite its utility, the ultrasound-guided neuraxial technique is still marginally used, even for patients with abnormal anatomy. Some experts attribute this to cost, a relatively high success rate without ultrasound, and a lack of technical expertise, which is often tied to formal education and regular practice. Several proponents of the ultrasound technique emphasize that proficiency requires practice on patients with normal spine anatomy, though this training may not be as challenging as once thought. This protocol was designed to help all providers learn the basics of lumbar spine anatomy and how to apply this knowledge clinically. Through a series of videos, we will provide step-by-step instructions for performing neuraxial ultrasonography and offer practical tips for troubleshooting in cases of difficult anatomy.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442452","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

Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy derived Force Maps.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-31 DOI: 10.3791/67784

Bastian Hartmann, Monica Nicolau, Raphael Reuten, Hauke Clausen-Schaumann

{"title":"Extracting the Young's Modulus of Native Murine Pulmonary Basement Membranes from Atomic Force Microscopy derived Force Maps.","authors":"Bastian Hartmann, Monica Nicolau, Raphael Reuten, Hauke Clausen-Schaumann","doi":"10.3791/67784","DOIUrl":"https://doi.org/10.3791/67784","url":null,"abstract":"Atomic force microscopy (AFM) allows the characterization of the mechanical properties of a sample with a spatial resolution of several tens of nanometers. Because mammalian cells sense and react to the mechanics of their immediate microenvironment, the characterization of biomechanical properties of tissues with high spatial resolution is crucial for understanding various developmental, homeostatic, and pathological processes. The basement membrane (BM), a roughly 100 - 400 nm thin extracellular matrix (ECM) substructure, plays a significant role in tumor progression and metastasis formation. Although determining Young's modulus of such a thin ECM substructure is challenging, biomechanical data of the BM provides fundamental new insights into how the BM affects cell behavior and, in addition, offers valuable diagnostic potential. Here, we present a visualized protocol for assessing BM mechanics in murine lung tissue, which is one of the major organs prone to metastasis. We describe an efficient workflow for determining the Young's modulus of the BM, which is located between the endothelial and epithelial cell layers in lung tissue. The step-by-step instructions comprise murine lung tissue freezing, cryosectioning, and AFM force-map recording on tissue sections. Additionally, we provide a semi-automatic data analysis procedure using the CANTER Processing Toolbox, an in-house developed user-friendly AFM data analysis software. This tool enables automatic loading of recorded force maps, conversion of force versus piezo-extension curves to force versus indentation curves, computation of Young's moduli, and generation of Young's modulus maps. Finally, it shows how to determine and isolate Young's modulus values derived from the pulmonary BM through the use of a spatial filtering tool.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143441898","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

GC-MS Analysis of Deuterium Enrichment of Retinol in Serum and Estimation of Vitamin A Total Body Stores.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-31 DOI: 10.3791/67518

Gabriel Medoua Nama

{"title":"GC-MS Analysis of Deuterium Enrichment of Retinol in Serum and Estimation of Vitamin A Total Body Stores.","authors":"Gabriel Medoua Nama","doi":"10.3791/67518","DOIUrl":"https://doi.org/10.3791/67518","url":null,"abstract":"This method describes the determination of deuterium enrichment of retinol in serum and the estimation of vitamin A stores in the body. The process involves extracting retinol from 0.4 mL of serum using 0.5 mL of 0.85% saline solution, 100 µL of internal standard solution, and 5 mL of chloroform-methanol (2:1 v/v) solution. After centrifugation and removal of the lower chloroform layer, the mixture is dried under nitrogen and resuspended in 0.1 mL of ethanol, and the retinol fraction is separated from other constituents using an HPLC system equipped with a PE C18 column. The retinol fraction can be collected manually or with a fraction collector. Subsequently, the retinol fraction is dried under nitrogen and derivatized with O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 10% trimethylchlorosilane. Finally, labeled and non-labeled retinol isotopes are quantified using a GC-MS system equipped with a 19091z-431 HP-1 methyl siloxane capillary column, employing electron capture negative chemical ionization with helium as the carrier gas and methane as the ionization agent. The ratio of labeled to non-labeled retinol is then used in the Olson, Green, or mass balance equations to estimate vitamin A stores.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143441901","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

Minimally Invasive Isolated Limb Perfusion (MI-ILP) for Locally Advanced Melanomas and Sarcomas of the Extremity.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-31 DOI: 10.3791/66876

Anne Huibers, Andrew Wong, Ellen Krabbe, Per Carlson, Roger Olofsson Bagge

引用次数: 0

Cortical Bone Assessment Using Ultrasonic Guided Waves: A Reproducibility Study in a Healthy Population.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-31 DOI: 10.3791/66985

Baptiste Dubot, Maxime Deniau, Donatien Ramiandrisoa, Jean-Gabriel Minonzio

{"title":"Cortical Bone Assessment Using Ultrasonic Guided Waves: A Reproducibility Study in a Healthy Population.","authors":"Baptiste Dubot, Maxime Deniau, Donatien Ramiandrisoa, Jean-Gabriel Minonzio","doi":"10.3791/66985","DOIUrl":"https://doi.org/10.3791/66985","url":null,"abstract":"Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected to double between 2020 and 2050. Therefore, it is essential to detect patients at risk of fragility fracture at the population scale. The current golden standard is dual X-ray absorptiometry (DXA), providing the areal bone mineral density (aBMD). Ultrasonic devices, usually more portable and cheaper than X-ray devices, represent interesting DXA alternatives as screening tools. However, operator dependency is usually recognized as their main drawback. In this study, the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device is presented in detail. The dedicated ultrasonic probe is placed at the one-third distal radius of the non-dominant forearm using conventional coupling gel. The guided interface provides in quasi-real time (about 2 Hz) four parameters of interest: velocities of the first arriving signal (vFAS) and the A0 mode (vA0), cortical thickness (Ct.Th) and porosity (Ct.Po), as well as four quality parameters. The operator moves the probe slowly at the measurement site, carefully observing the feedback provided by the interface until finding a stable position and starting a series of 10 acquisitions. When at least four consistent series are obtained, the measurement ends, and an automatic report is generated. The measurement usually takes about 5 min to complete. To determine the robustness of this protocol, a reproducibility study was conducted among 3 operators (one expert and two novices) and 14 healthy participants (6 women, 8 men, 21-53 years old). The intraclass correlation coefficients (ICC) were found good for vA0 (0.76), Ct.Po (0.80) or excellent for Ct.Th (0.87) and vFAS (0.91). The standard deviations were found to be less than 10% of the total ranges in clinical practice.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143442585","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

Fine Adjustment of Caenorhabditis elegans Orientation on Channeled Agar Pads for Imaging Neuroregeneration.

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-31 DOI: 10.3791/67811

Tina Thuy N Nguyen Hoang, Chirayu P Sanganeria, Samuel H Chung

{"title":"Fine Adjustment of Caenorhabditis elegans Orientation on Channeled Agar Pads for Imaging Neuroregeneration.","authors":"Tina Thuy N Nguyen Hoang, Chirayu P Sanganeria, Samuel H Chung","doi":"10.3791/67811","DOIUrl":"https://doi.org/10.3791/67811","url":null,"abstract":"Caenorhabditis elegans is a model organism widely used for studying biological processes. Its transparency and small size make it ideal for imaging tissues, cells, and subcellular structures. Traditional flat agar pads for imaging C. elegans limit control over the animal's orientation, restricting views primarily to lateral perspectives. This limitation complicates the visualization of dorsal-ventral structures and reduces image clarity, especially in older animals with increased pigmentation and larger diameters. To overcome these challenges, we developed channeled agarose pads that allow precise control of animal orientation. These channels enable researchers to rotate and fix C. elegans in specified positions, facilitating the simultaneous imaging of multiple structures and improving image resolution by bringing target cells closer to the microscope objective. This is particularly useful for imaging regenerated neuronal fibers after surgery, which may grow in directions difficult to capture with traditional flat agar pads. This method is accessible, as fabricating channeled agar pads requires the same time and materials as flat pads, making it a practical option for most laboratories.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143441899","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