Jove-Journal of Visualized Experiments最新文献

Erratum: A Mouse Model of Hemorrhagic Transformation Induced by Acute Hyperglycemia Combined with Transient Focal Ischemia. 更正：急性高血糖合并短暂局灶性缺血致出血转化的小鼠模型。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-06 DOI: 10.3791/6615

Yaojian Sun, Changlong Leng, Kang Ma, Wei Liu

引用次数: 0

ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils. 小鼠骨髓中性粒细胞的ATAC-Seq文库制备。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-03 DOI: 10.3791/67490

Ju Zhang, Shuai Tong, Siyuan Liang, Fangyuan Li, Can Zhang, Nan Ding, Yu Hao

{"title":"ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils.","authors":"Ju Zhang, Shuai Tong, Siyuan Liang, Fangyuan Li, Can Zhang, Nan Ding, Yu Hao","doi":"10.3791/67490","DOIUrl":"https://doi.org/10.3791/67490","url":null,"abstract":"Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and understanding epigenomic regulation. Neutrophils, as a crucial leukocyte type in immune responses, undergo substantial chromatin architectural changes during differentiation and activation, which significantly impact the gene expression necessary for their functions. ATAC-seq has been instrumental in uncovering key transcription factors in neutrophil maturation, revealing pathogen-specific epigenomic signatures, and identifying therapeutic targets for autoimmune diseases. However, neutrophils' sensitivity to the external milieu complicates high-quality ATAC-seq data production. Here, we propose a scalable protocol for preparing ATAC-seq libraries from rodent bone marrow-derived neutrophils, featuring improved immunomagnetic separation to ensure optimal cell viability and high-quality libraries. The vital elements impacting the library quality and optimization principles for methodological extension are discussed in detail. This protocol will support the researchers who are willing to study the chromatin architecture and epigenomic reprogramming of neutrophils, advancing studies in basic and clinical immunology.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143013563","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

Single-Animal, Single-Tube RNA Extraction for Comparison of Relative Transcript Levels via qRT-PCR in the Tardigrade Hypsibius exemplaris. 单动物，单管RNA提取，通过qRT-PCR比较缓步动物样土潜虫的相对转录水平。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-03 DOI: 10.3791/66935

Molly J Kirk, Chaoming Xu, Jonathan Paules, Joel H Rothman

{"title":"Single-Animal, Single-Tube RNA Extraction for Comparison of Relative Transcript Levels via qRT-PCR in the Tardigrade Hypsibius exemplaris.","authors":"Molly J Kirk, Chaoming Xu, Jonathan Paules, Joel H Rothman","doi":"10.3791/66935","DOIUrl":"https://doi.org/10.3791/66935","url":null,"abstract":"The tardigrade Hypsibius exemplaris is an emerging model organism renowned for its ability to survive environmental extremes. To explore the molecular mechanisms and genetic basis of such extremotolerance, many studies rely on RNA-sequencing (RNA-seq), which can be performed on populations ranging from large cohorts to individual animals. Reverse transcription polymerase chain reaction (RT-PCR) and RNA interference (RNAi) are subsequently used to confirm RNA-seq findings and assess the genetic requirements for candidate genes, respectively. Such studies require an efficient, accurate, and affordable method for RNA extraction and measurement of relative transcript levels by quantitative RT-PCR (qRT-PCR). This work presents an efficient single-tardigrade, single-tube RNA extraction method (STST) that not only reliably isolates RNA from individual tardigrades but also reduces the required time and cost for each extraction. This RNA extraction method yields quantities of cDNA that can be used to amplify and detect multiple transcripts by quantitative PCR (qRT-PCR). The method is validated by analyzing dynamic changes in the expression of genes encoding two heat-shock-regulated proteins, Heat-Shock Protein 70 β2 (HSP70 β2) and Heat-Shock Protein 90α (HSP90α), making it possible to assess their relative expression levels in heat-exposed individuals using qRT-PCR. STST effectively complements existing bulk and single tardigrade RNA extraction methods, permitting rapid and affordable examination of individual tardigrade transcriptional levels by qRT-PCR.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143014432","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 Different Connection Modes of Electroacupuncture on Electrocardiogram and Nerve Discharge in Rats. 电针不同连接方式对大鼠心电图及神经放电的影响。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-03 DOI: 10.3791/65945

Yemao Chai, Mengwei Guo, Xueling Kang, Xiaying Li, Xiaoxuan Ren

{"title":"Effects of Different Connection Modes of Electroacupuncture on Electrocardiogram and Nerve Discharge in Rats.","authors":"Yemao Chai, Mengwei Guo, Xueling Kang, Xiaying Li, Xiaoxuan Ren","doi":"10.3791/65945","DOIUrl":"https://doi.org/10.3791/65945","url":null,"abstract":"Electroacupuncture (EA) is one of the most commonly used methods in acupuncture and has a good effect on pain, depression, sensory movement disorders, and other diseases. The effectiveness of EA is influenced by many factors, such as the accuracy of acupoint selection, the duration and course of EA treatment, and EA parameters. However, it has rarely been discussed whether the positive and negative electrodes of the EA instrument with acupoints at different locations and distances have an effect on the curative effect. In this experiment, we observed the effects of connecting the EA instrument to acupoints at different positions and distances on ECG and vague nerve discharge in rats and preliminarily explained whether the electric field formed by different modes of EA connection has an effect on the function of the body. The connection modes of EA in this experiment included the same acupoints on both sides of the body, the same meridian or different meridian acupoints on the same side of the body, and two needles in the same acupoint area. The results showed that when the positive and negative poles were connected to the acupoints on both sides of the body, the recording of ECG and vagal nerve activity was disturbed (the same fore and hind limbs); when the acupoints were connected on the same side of the body, the smaller the distance between the two needles, the smaller the effect on ECG and vagal nerve activity recording, and the effect increased with increasing current; when the acupoints were in the same acupoint area, the recording of ECG and vagal nerve activity was not disturbed if the two needles did not form a short circuit.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143013950","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

Breeding by Design for Functional Rice with Genome Editing Technologies. 利用基因组编辑技术设计功能水稻育种。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-03 DOI: 10.3791/67336

Chunxue Yan, Huicong Meng, Yanxin Pei, Wei Sun, Jinshan Zhang

{"title":"Breeding by Design for Functional Rice with Genome Editing Technologies.","authors":"Chunxue Yan, Huicong Meng, Yanxin Pei, Wei Sun, Jinshan Zhang","doi":"10.3791/67336","DOIUrl":"https://doi.org/10.3791/67336","url":null,"abstract":"The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143013622","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

Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke. 用免疫荧光评价大鼠缺血性脑卒中模型中的细胞死亡信号。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-03 DOI: 10.3791/67606

Hengshu Chen, Kexin Chen, Xing Hua, Simiao Wu, Weihong He

{"title":"Evaluating Cell Death Signaling by Immunofluorescence in a Rat Model of Ischemic Stroke.","authors":"Hengshu Chen, Kexin Chen, Xing Hua, Simiao Wu, Weihong He","doi":"10.3791/67606","DOIUrl":"https://doi.org/10.3791/67606","url":null,"abstract":"Stroke is a leading cause of death and disability worldwide. Most cases of stroke are ischemic and result from the occlusion of the middle cerebral artery (MCA). Current pharmacological approaches for the treatment of ischemic stroke are limited; therefore, novel therapies providing effective neuroprotection against ischemic injury following stroke are urgently needed. In the brain tissue following ischemic stroke, the area of the ischemic penumbra is salvageable but at risk of progressing to irreversible damage. The penumbral area surrounding the infarcted core is a conceptual target for neuroprotection. Since the blood flow is partially maintained in the penumbral area, neuronal and non-neuronal cells in this area transiently survive after stroke, and these cells that are still viable may be rescued by appropriate medical interventions. Understanding the pathophysiology of the penumbra is important in the development of neuroprotective therapies because the cell death pathways activated in the ischemic penumbra may indicate therapeutic targets, such as RUNX1 and cathepsins. These protein targets functioning as mediators of programmed cell death can be further exploited in translational research. With moderate size and similarity to the human brain, the in vivo rat model of MCA occlusion (MCAO) mimics the human ischemic stroke and offers an applicable tool to investigate the penumbral pathology, examine the cell death signaling, and evaluate the effects of potential targets in the context of MCAO. Here, we describe how to induce MCAO in rats and how to perform immunofluorescence staining for the detection of cell death signaling in the rat brain following MCAO.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143014161","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

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads. 利用磁珠操纵发育中的斑马鱼心脏中的机械力。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-03 DOI: 10.3791/67604

Christina Vagena-Pantoula, Hajime Fukui, Julien Vermot

引用次数: 0

Quantifying Pulmonary Microvascular Density in Mice Across Lobules. 定量小鼠肺小叶微血管密度。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-03 DOI: 10.3791/66681

Chunyan Li, Zhu Wang, Junrong Du, Tao Jia

{"title":"Quantifying Pulmonary Microvascular Density in Mice Across Lobules.","authors":"Chunyan Li, Zhu Wang, Junrong Du, Tao Jia","doi":"10.3791/66681","DOIUrl":"https://doi.org/10.3791/66681","url":null,"abstract":"The abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction and is deeply linked to vascular wall integrity, blood flow regulation, and gas exchange. In murine models, lung lobes exhibit significant differences in size, shape, location, and vascularization, yet existing methods lack consideration for these variations when quantifying microvascular density. This limitation hinders the comprehensive study of lung microvascular dysfunction and the potential remodeling of microvasculature circulation across different lobules. Our protocol addresses this gap by employing two sectioning methods to quantify pulmonary microvascular density changes, leveraging the size, shape, and distribution of airway branches across distinct lobes in mice. We then utilize Isolectin B4 (IB4) staining to label lung microvascular endothelial cells on different slices, followed by unbiased microvascular density analysis using the freely available software ImageJ. The results presented here highlight varying degrees of microvascular density changes across lung lobules with aging, comparing young and old mice. This protocol offers a straightforward and cost-effective approach for unbiased quantification of pulmonary microvascular density, facilitating research on both physiological and pathological aspects of lung microvasculature.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143014415","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

Generation of Human Chimeric Antigen Receptor Regulatory T Cells. 人嵌合抗原受体调节性T细胞的产生。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2025-01-03 DOI: 10.3791/67200

Russell W Cochrane, Rob A Robino, Leonardo M R Ferreira

{"title":"Generation of Human Chimeric Antigen Receptor Regulatory T Cells.","authors":"Russell W Cochrane, Rob A Robino, Leonardo M R Ferreira","doi":"10.3791/67200","DOIUrl":"https://doi.org/10.3791/67200","url":null,"abstract":"Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs' immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 215","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143014162","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

Surgical Transplantation of Tumor Cells into the Spinal Cord of Mice. 肿瘤细胞在小鼠脊髓内的外科移植。

IF 1.2 4区综合性期刊

Jove-Journal of Visualized Experiments Pub Date : 2024-12-27 DOI: 10.3791/67269

Xun Mingjuan, Lin Han, Yu Caiyuan, Pang Bo, Wang Yongzhi, Yan Jun

引用次数: 0