Valeria Di Gioia, Jan Zamporlini, Rebecca Vadalà, Elena Parmigiani, Beatrice Bodega, Federica Marasca
Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience. In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution. Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different contexts.
各种核过程(如转录控制)都发生在被称为病灶的离散结构中,通过免疫荧光技术可以分辨出这些病灶。通过显微镜研究这些病灶在不同细胞条件下的动态变化,可以深入了解细胞特性和功能的分子机制。然而,在不同类型的细胞中进行免疫荧光检测,并评估这些病灶的组装、扩散和分布变化,却面临着诸多挑战。这些挑战包括样品制备的复杂性、成像数据分析参数的确定以及大量数据的管理。此外,现有的成像工作流程通常是为熟练用户量身定制的,因此限制了更多人的使用。在本研究中,我们介绍了一种优化的免疫荧光方案,专门用于研究不同人类原代 T 细胞类型中的核蛋白,该方案可针对任何感兴趣的蛋白质和细胞类型进行定制。此外,我们还介绍了一种无偏量化蛋白质染色的方法,无论它们是形成明显的病灶还是呈现弥散的核分布。我们提出的方法提供了从细胞染色到分析的全面指导,利用 Jython 开发的半自动化管道,可在 Fiji 中执行。此外,我们还提供了一个用户友好型 Python 脚本,以简化数据管理,该脚本可在 Google Colab 笔记本上公开访问。我们的方法在对不同背景下具有不同核组织模式的蛋白质进行高信息量的免疫荧光分析方面证明了其有效性。
{"title":"A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types.","authors":"Valeria Di Gioia, Jan Zamporlini, Rebecca Vadalà, Elena Parmigiani, Beatrice Bodega, Federica Marasca","doi":"10.3791/66874","DOIUrl":"https://doi.org/10.3791/66874","url":null,"abstract":"<p><p>Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience. In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution. Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different contexts.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141617454","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}
Alexander Wilson, Varuna Chaudhary, Ela Chakkrapani, Mary O'Dea, Jonathan Davis
Infants at risk of HIE require early identification and initiation of therapeutic hypothermia (TH). Earlier treatment with TH is associated with better outcomes. aEEG is frequently used when making the decision whether to commence TH. As this is often limited to tertiary centers, TH may be delayed if the infant requires transport to a center that provides it. We aimed to provide a method for the application of amplitude-integrated electroencephalogram (aEEG) and to determine the feasibility of acquiring clinically meaningful information during transport. All infants ≥35 weeks, at risk of HIE at referral, were eligible for inclusion. Scalp electrodes were placed in the C3-C4; P3-P4 position on the infant's scalp and connected to the aEEG amplifier. The aEEG amplifier was, in turn, connected to a clinical tablet computer with EEG software to collect and analyze aEEG information. Recordings were reviewed by the chief principal investigator and two independent reviewers (blinded) for background trace and artifact. Predefined criteria for data quality were set to movement artifacts and software impedance notifications. Surveys were completed by healthcare staff and parents for acceptability and ease of use.
{"title":"Amplitude-Integrated EEG in Infants at Risk of Hypoxic-Ischemic Encephalopathy: A Feasibility Study in Road and Air Transport in Western Australia.","authors":"Alexander Wilson, Varuna Chaudhary, Ela Chakkrapani, Mary O'Dea, Jonathan Davis","doi":"10.3791/66825","DOIUrl":"https://doi.org/10.3791/66825","url":null,"abstract":"<p><p>Infants at risk of HIE require early identification and initiation of therapeutic hypothermia (TH). Earlier treatment with TH is associated with better outcomes. aEEG is frequently used when making the decision whether to commence TH. As this is often limited to tertiary centers, TH may be delayed if the infant requires transport to a center that provides it. We aimed to provide a method for the application of amplitude-integrated electroencephalogram (aEEG) and to determine the feasibility of acquiring clinically meaningful information during transport. All infants ≥35 weeks, at risk of HIE at referral, were eligible for inclusion. Scalp electrodes were placed in the C3-C4; P3-P4 position on the infant's scalp and connected to the aEEG amplifier. The aEEG amplifier was, in turn, connected to a clinical tablet computer with EEG software to collect and analyze aEEG information. Recordings were reviewed by the chief principal investigator and two independent reviewers (blinded) for background trace and artifact. Predefined criteria for data quality were set to movement artifacts and software impedance notifications. Surveys were completed by healthcare staff and parents for acceptability and ease of use.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555769","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}
Brian T Nguyen, Nora M Chapman, John C Mullican, Kristen M Drescher
All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).
奈格勒氏滋养体的所有核糖体基因都保存在一个封闭的环状染色体外核糖体 DNA(rDNA)含元(CERE)中。虽然人们对 CERE 知之甚少,但对三种奈格勒氏滋养体的完整基因组序列分析清楚地表明,核基因组中没有 rDNA 单列子。此外,在 N. gruberi CERE 中绘制了一个单一的 DNA 复制源,支持了 CERE 独立于核基因组进行复制的假设。CERE 的这一特性表明,有可能利用改造的 CERE 将外来蛋白质引入奈格勒氏滋养体。作为探索在奈格勒氏滋养体中使用 CERE 作为载体的第一步,我们制定了一套方案,用克隆到 pGEM7zf+ (pGRUB)中的奈格勒氏滋养体 CERE 分子克隆转染奈格勒氏滋养体。转染后,pGRUB 可在 N. gruberi 滋养体中检测到至少 7 个传代以及包囊和外包囊。作为对照,滋养体转染了不含 N. gruberi 序列的骨干载体 pGEM7zf+(pGEM)。pGEM 在转染到 N. gruberi 滋养体后的第一次传代后就检测不到了,这表明它无法在真核生物体内复制。这些研究描述了奈格拉滋养体的转染方案,并证明 pGRUB 中的细菌质粒序列不会抑制转染 CERE 克隆的成功转染和复制。此外,该转染方案对于了解 CERE 在滋养体中复制的最小驱动序列以及确定非核糖体序列(NRS)中的调控区域至关重要。
{"title":"Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites.","authors":"Brian T Nguyen, Nora M Chapman, John C Mullican, Kristen M Drescher","doi":"10.3791/66726","DOIUrl":"https://doi.org/10.3791/66726","url":null,"abstract":"<p><p>All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555779","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}
Maria Magdalena Mair, Ruth Schröcksnadel, Kurt Stephan, Josef Seebacher, Philipp Zelger, Franz Muigg, Timo Gottfried, Annett Franke-Trieger, Joachim Schmutzhard
Measuring the electrically evoked stapedius reflex during the fitting of cochlear implants (CIs) provides a reliable estimation of maximum comfort levels, resulting in the programming of the CI with high hearing comfort and good speech understanding. Detection of the stapedius reflex and the required stimulation level on each implant channel is already being performed during surgery, whereby intraoperative stapedius reflexes are observed through the surgical microscope. Intraoperative stapedius reflex detection is both an indicator that the auditory nerve is responding to electrical stimulation up to the brainstem and a test for the ability to perform postoperative stapedius reflex measurements. Postoperative stapedius reflex thresholds can be used to estimate upper stimulation levels in the CI fitting process. In particular, in children or patients unable to provide feedback on loudness perception, this method avoids inadequate stimulation with the CI, which can result in poor hearing performance. In addition, overstimulation can be avoided, which could even lead to refusal to use the device.
在安装人工耳蜗(CI)时,通过测量电诱发的镫骨反射,可以可靠地估计最大舒适度,从而使人工耳蜗的编程具有较高的听力舒适度和良好的言语理解能力。在手术过程中,通过手术显微镜观察术中镫骨反射,并确定每个植入通道所需的刺激水平。术中镫骨反射检测既是听神经对直至脑干的电刺激做出反应的指标,也是对术后镫骨反射测量能力的测试。术后镫骨反射阈值可用于估计 CI 装配过程中的上限刺激水平。特别是对于无法提供响度感知反馈的儿童或患者,这种方法可以避免 CI 刺激不足,从而导致听力表现不佳。此外,还可以避免过度刺激,因为过度刺激甚至会导致患者拒绝使用设备。
{"title":"Electrically Evoked Stapedius Reflex Measurements in Cochlear Implantation and its Application in the Postoperative Fitting Process.","authors":"Maria Magdalena Mair, Ruth Schröcksnadel, Kurt Stephan, Josef Seebacher, Philipp Zelger, Franz Muigg, Timo Gottfried, Annett Franke-Trieger, Joachim Schmutzhard","doi":"10.3791/66526","DOIUrl":"https://doi.org/10.3791/66526","url":null,"abstract":"<p><p>Measuring the electrically evoked stapedius reflex during the fitting of cochlear implants (CIs) provides a reliable estimation of maximum comfort levels, resulting in the programming of the CI with high hearing comfort and good speech understanding. Detection of the stapedius reflex and the required stimulation level on each implant channel is already being performed during surgery, whereby intraoperative stapedius reflexes are observed through the surgical microscope. Intraoperative stapedius reflex detection is both an indicator that the auditory nerve is responding to electrical stimulation up to the brainstem and a test for the ability to perform postoperative stapedius reflex measurements. Postoperative stapedius reflex thresholds can be used to estimate upper stimulation levels in the CI fitting process. In particular, in children or patients unable to provide feedback on loudness perception, this method avoids inadequate stimulation with the CI, which can result in poor hearing performance. In addition, overstimulation can be avoided, which could even lead to refusal to use the device.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555771","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}
Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, development, size, and transparency. Conversely, dysfunctional LECs can lead to cataract formation and posterior capsule opacification (PCO). Consequently, establishing a robust primary LEC culture system is important to researchers engaged in lens development, biochemistry, cataract therapeutics, and PCO prevention. However, cultivating primary LECs has long presented challenges due to their limited availability, slow proliferation rate, and delicate nature. This study addresses these hurdles by presenting a comprehensive protocol for primary LEC culture. The protocol encompasses essential steps such as the formulation of an optimized culture medium, precise isolation of lens capsules, trypsinization techniques, subculture procedures, harvest protocols, and guidelines for storage and shipment. Throughout the culture process, cell morphology was monitored using phase-contrast microscopy. To confirm the authenticity of the cultured LECs, immunofluorescence assays were conducted to detect the presence and subcellular distribution of critical lens proteins, namely αA- and γ-crystallins. This detailed protocol equips researchers with a valuable resource for cultivating and characterizing primary LECs, enabling advancements in our comprehension of lens biology and the development of therapeutic strategies for lens-related disorders.
{"title":"Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization.","authors":"Yu Yu, Jinmin Zhang, Hongli Wu","doi":"10.3791/65912","DOIUrl":"10.3791/65912","url":null,"abstract":"<p><p>Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, development, size, and transparency. Conversely, dysfunctional LECs can lead to cataract formation and posterior capsule opacification (PCO). Consequently, establishing a robust primary LEC culture system is important to researchers engaged in lens development, biochemistry, cataract therapeutics, and PCO prevention. However, cultivating primary LECs has long presented challenges due to their limited availability, slow proliferation rate, and delicate nature. This study addresses these hurdles by presenting a comprehensive protocol for primary LEC culture. The protocol encompasses essential steps such as the formulation of an optimized culture medium, precise isolation of lens capsules, trypsinization techniques, subculture procedures, harvest protocols, and guidelines for storage and shipment. Throughout the culture process, cell morphology was monitored using phase-contrast microscopy. To confirm the authenticity of the cultured LECs, immunofluorescence assays were conducted to detect the presence and subcellular distribution of critical lens proteins, namely αA- and γ-crystallins. This detailed protocol equips researchers with a valuable resource for cultivating and characterizing primary LECs, enabling advancements in our comprehension of lens biology and the development of therapeutic strategies for lens-related disorders.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11259111/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555775","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ana Luísa Graça, Niklas Kroner-Weigl, Viviana Reyes Alcaraz, Sigrid Müller-Deubert, Maximilian Rudert, Denitsa Docheva
Tendons and ligaments (T/L) are strong hierarchically organized structures uniting the musculoskeletal system. These tissues have a strictly arranged collagen type I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGFß is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.
{"title":"Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts.","authors":"Ana Luísa Graça, Niklas Kroner-Weigl, Viviana Reyes Alcaraz, Sigrid Müller-Deubert, Maximilian Rudert, Denitsa Docheva","doi":"10.3791/66047","DOIUrl":"https://doi.org/10.3791/66047","url":null,"abstract":"<p><p>Tendons and ligaments (T/L) are strong hierarchically organized structures uniting the musculoskeletal system. These tissues have a strictly arranged collagen type I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGFß is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555770","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}
Michael Turk, Benjamin Catanese, Brice Lefler, Matthew A Sparks, Yuriy S Bronshteyn, Amber Bowman
A range of conditions involving the kidneys and urinary bladder can cause organ-threatening complications that are preventable if diagnosed promptly with diagnostic imaging. Common imaging modalities include either computed tomography or diagnostic ultrasound. Traditionally, ultrasound of the kidney-genitourinary system has required consultative teams consisting of a sonographer performing image acquisition and a radiologist performing image interpretation. However, diagnostic point-of-care ultrasound (POCUS) has recently emerged as a useful tool to troubleshoot acute kidney injury at the bedside. Studies have shown that non-radiologists can be trained to perform diagnostic POCUS of the kidneys and bladder with high accuracy for a set number of important conditions. Currently, diagnostic POCUS of the kidney-genitourinary system remains underused in actual clinical practice. This is likely because image acquisition for this organ system is unfamiliar to most clinicians in specialties that encounter acute kidney injury, including primary care, emergency medicine, intensive care, anesthesiology, nephrology, and urology. To address this multi-specialty educational gap, this narrative review was developed by a multi-disciplinary group to provide a specialty-agnostic framework for kidney-genitourinary POCUS image acquisition: indications/contraindications, patient positioning, transducer selection, acquisition sequence, and exam limitations. Finally, we describe foundational concepts in kidney-genitourinary ultrasound image interpretation, including key abnormal findings that every bedside clinician performing this modality should know.
{"title":"Point-of-Care Kidney and Genitourinary Ultrasound in Adults: Image Acquisition.","authors":"Michael Turk, Benjamin Catanese, Brice Lefler, Matthew A Sparks, Yuriy S Bronshteyn, Amber Bowman","doi":"10.3791/66802","DOIUrl":"https://doi.org/10.3791/66802","url":null,"abstract":"<p><p>A range of conditions involving the kidneys and urinary bladder can cause organ-threatening complications that are preventable if diagnosed promptly with diagnostic imaging. Common imaging modalities include either computed tomography or diagnostic ultrasound. Traditionally, ultrasound of the kidney-genitourinary system has required consultative teams consisting of a sonographer performing image acquisition and a radiologist performing image interpretation. However, diagnostic point-of-care ultrasound (POCUS) has recently emerged as a useful tool to troubleshoot acute kidney injury at the bedside. Studies have shown that non-radiologists can be trained to perform diagnostic POCUS of the kidneys and bladder with high accuracy for a set number of important conditions. Currently, diagnostic POCUS of the kidney-genitourinary system remains underused in actual clinical practice. This is likely because image acquisition for this organ system is unfamiliar to most clinicians in specialties that encounter acute kidney injury, including primary care, emergency medicine, intensive care, anesthesiology, nephrology, and urology. To address this multi-specialty educational gap, this narrative review was developed by a multi-disciplinary group to provide a specialty-agnostic framework for kidney-genitourinary POCUS image acquisition: indications/contraindications, patient positioning, transducer selection, acquisition sequence, and exam limitations. Finally, we describe foundational concepts in kidney-genitourinary ultrasound image interpretation, including key abnormal findings that every bedside clinician performing this modality should know.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555776","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}
The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.
{"title":"Isolation of Mouse Pancreatic Endothelial Cells.","authors":"Alonso Tapia, Naseeb Kaur Malhi, Xuejing Liu, Muxi Chen, Zhen Bouman Chen","doi":"10.3791/66690","DOIUrl":"https://doi.org/10.3791/66690","url":null,"abstract":"<p><p>The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555774","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}
The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically limited by the number of fluorescent parameters that can be imaged at a single time. Most immune cell subsets have been defined using flow cytometry by using complex combinations of protein markers, often requiring four or more parameters to conclusively identify, which is beyond the capabilities of most conventional microscopes. As flow cytometry dissociates tissues and loses spatial information, there is a need for techniques that can retain spatial information while interrogating the roles of complex cell types. These issues are addressed here by creating a method for expanding the number of fluorescent parameters that can be imaged by collecting the signals of spectrally overlapping fluorophores and using spectral unmixing to separate the signals of each individual fluorophore. These images are then processed using an analysis pipeline to take high-parameter histology images and extract single cells from these images so that the unique fluorescent properties of each cell can be analyzed at a single-cell level. Using flow cytometry-like gating strategies, cells can then be profiled into subsets and mapped back onto the histology sections to not only quantify their abundance, but also establish how they interact with the tissue environment. Overall, the simplicity and potential of using histoflow cytometry to study complex immune populations in histology sections is demonstrated.
{"title":"Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry.","authors":"Rajiv W Jain, David A Elliott, V Wee Yong","doi":"10.3791/66889","DOIUrl":"https://doi.org/10.3791/66889","url":null,"abstract":"<p><p>The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically limited by the number of fluorescent parameters that can be imaged at a single time. Most immune cell subsets have been defined using flow cytometry by using complex combinations of protein markers, often requiring four or more parameters to conclusively identify, which is beyond the capabilities of most conventional microscopes. As flow cytometry dissociates tissues and loses spatial information, there is a need for techniques that can retain spatial information while interrogating the roles of complex cell types. These issues are addressed here by creating a method for expanding the number of fluorescent parameters that can be imaged by collecting the signals of spectrally overlapping fluorophores and using spectral unmixing to separate the signals of each individual fluorophore. These images are then processed using an analysis pipeline to take high-parameter histology images and extract single cells from these images so that the unique fluorescent properties of each cell can be analyzed at a single-cell level. Using flow cytometry-like gating strategies, cells can then be profiled into subsets and mapped back onto the histology sections to not only quantify their abundance, but also establish how they interact with the tissue environment. Overall, the simplicity and potential of using histoflow cytometry to study complex immune populations in histology sections is demonstrated.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555773","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}
Thien N M Doan, Daisuke Maruyama, Xiaoli Tian, Arun Prakash
Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous distant organ systems. Short-chain fatty acids (SCFA) are one class of metabolites produced by gut microbes during the fermentation of indigestible dietary fiber. They are now recognized as important contributors to how the gut microbiome influences extra-intestinal organ systems via the gut-lung, gut-brain, and other gut-organ axes throughout the host. SCFAs are absorbed from the colon, through intestinal tissue, into the portal vein (PV). They then pass through the liver, and are consumed in various organs such as the brain, muscle, adipose tissue, and lungs. SCFAs are most easily measured in the expelled fecal material however, more accurate measurements have been obtained from intra-colonic fecal contents. Here we propose that sampling PV and systemic circulating plasma of a single subject may be preferable for studying the absorption, transport, and systemic levels of SCFAs in mice. We present a new technique for efficient blood sampling from the PV and inferior vena cava (IVC) that allows for the collection of relatively large volumes of blood from the portal and systemic circulations. This is accomplished by ligating the PV, thereby allowing for the dilation or enlargement of the PV as it backfills from the mesenteric veins that drain into it. Using this method, we were able to improve the rate of successful collection as well as the total amount of blood collected (up to 0.3 mL from IVC and 0.5 mL from PV).
{"title":"Sequential Blood Collection from Inferior Vena Cava Followed by Portal Vein to Evaluate Gut Microbial Metabolites in Mice.","authors":"Thien N M Doan, Daisuke Maruyama, Xiaoli Tian, Arun Prakash","doi":"10.3791/66673","DOIUrl":"https://doi.org/10.3791/66673","url":null,"abstract":"<p><p>Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous distant organ systems. Short-chain fatty acids (SCFA) are one class of metabolites produced by gut microbes during the fermentation of indigestible dietary fiber. They are now recognized as important contributors to how the gut microbiome influences extra-intestinal organ systems via the gut-lung, gut-brain, and other gut-organ axes throughout the host. SCFAs are absorbed from the colon, through intestinal tissue, into the portal vein (PV). They then pass through the liver, and are consumed in various organs such as the brain, muscle, adipose tissue, and lungs. SCFAs are most easily measured in the expelled fecal material however, more accurate measurements have been obtained from intra-colonic fecal contents. Here we propose that sampling PV and systemic circulating plasma of a single subject may be preferable for studying the absorption, transport, and systemic levels of SCFAs in mice. We present a new technique for efficient blood sampling from the PV and inferior vena cava (IVC) that allows for the collection of relatively large volumes of blood from the portal and systemic circulations. This is accomplished by ligating the PV, thereby allowing for the dilation or enlargement of the PV as it backfills from the mesenteric veins that drain into it. Using this method, we were able to improve the rate of successful collection as well as the total amount of blood collected (up to 0.3 mL from IVC and 0.5 mL from PV).</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141555777","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}