Current protocols in mouse biology最新文献

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The Kinetics of Intestinal Permeability in a Mouse Model of Traumatic Brain Injury 创伤性脑损伤小鼠模型肠通透性动力学
Current protocols in mouse biology Pub Date : 2020-12-02 DOI: 10.1002/cpmo.86
Jessica L. Weaver
{"title":"The Kinetics of Intestinal Permeability in a Mouse Model of Traumatic Brain Injury","authors":"Jessica L. Weaver","doi":"10.1002/cpmo.86","DOIUrl":"10.1002/cpmo.86","url":null,"abstract":"<p>Traumatic brain injury (TBI) is a leading cause of morbidity and mortality among trauma patients. Increased intestinal permeability plays an important role in the inflammatory process that accompanies TBI, and therapies that prevent this permeability change may improve outcomes in TBI patients. Different animal models have been developed to test permeability changes, but there has been no agreement on when permeability should be tested after TBI. Here, we describe a method for creating the TBI mouse model and for measuring intestinal permeability. We also detail our permeability measurements at different time points after TBI to help guide future experimental design. The TBI is made using a controlled cortical impact model with the cortical impactor set to speed 6 m/s, depth 3 mm, dwell time 0.2 s, and tip size 3 mm to produce a severe TBI. Permeability is measured at 2, 4, 6, and 24 hr after TBI by removing a piece of terminal ileum, tying the ends, filling the lumen with FITC-labeled dextran, and then measuring how much of the dextran moves into the surrounding solution bath over time using a fluorescent plate reader. Our results show that peak permeability occurs between 4 and 6 hr after TBI. We recommend that future experiments incorporate permeability measurements 4 to 6 hr after TBI in order to take advantage of this peak permeability. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Mouse CCI traumatic brain injury model and intestinal permeability measurement</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.86","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38665380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
An Ex Vivo Acid Injury and Repair (AIR) Model Using Precision-Cut Lung Slices to Understand Lung Injury and Repair 一个体外酸损伤和修复(AIR)模型使用精确切割的肺切片来了解肺损伤和修复
Current protocols in mouse biology Pub Date : 2020-11-20 DOI: 10.1002/cpmo.85
Sally Yunsun Kim, Róisín Mongey, Mark Griffiths, Matthew Hind, Charlotte H. Dean
{"title":"An Ex Vivo Acid Injury and Repair (AIR) Model Using Precision-Cut Lung Slices to Understand Lung Injury and Repair","authors":"Sally Yunsun Kim,&nbsp;Róisín Mongey,&nbsp;Mark Griffiths,&nbsp;Matthew Hind,&nbsp;Charlotte H. Dean","doi":"10.1002/cpmo.85","DOIUrl":"10.1002/cpmo.85","url":null,"abstract":"<p>Recent advances in cell culture models like air‒liquid interface culture and ex vivo models such as organoids have advanced studies of lung biology; however, gaps exist between these models and tools that represent the complexity of the three-dimensional environment of the lung. Precision-cut lung slices (PCLS) mimic the in vivo environment and bridge the gap between in vitro and in vivo models. We have established the acid injury and repair (AIR) model where a spatially restricted area of tissue is injured using drops of HCl combined with Pluronic gel. Injury and repair are assessed by immunofluorescence using robust markers, including Ki67 for cell proliferation and prosurfactant protein C for alveolar type 2/progenitor cells. Importantly, the AIR model enables the study of injury and repair in mouse lung tissue without the need for an initial in vivo injury, and the results are highly reproducible. Here, we present detailed protocols for the generation of PCLS and the AIR model. We also describe methods to analyze and quantify injury in AIR-PCLS by immunostaining with established early repair markers and fluorescence imaging. This novel ex vivo model is a versatile tool for studying lung cell biology in acute lung injury and for semi-high-throughput screening of potential therapeutics. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Generation of precision-cut lung slices</p><p><b>Basic Protocol 2</b>: The acid injury and repair model</p><p><b>Basic Protocol 3</b>: Analysis of AIR-PCLS: Immunostaining and imaging</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.85","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38634733","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 5
Tackling the Mouse-on-Mouse Problem in Cochlear Immunofluorescence: A Simple Double-Blocking Protocol for Immunofluorescent Labeling of Murine Cochlear Sections with Primary Mouse Antibodies 解决耳蜗免疫荧光中小鼠对小鼠的问题:用小鼠一抗免疫荧光标记小鼠耳蜗切片的简单双阻断方案
Current protocols in mouse biology Pub Date : 2020-10-02 DOI: 10.1002/cpmo.84
Mohamed Bassiouni, Katharina Stölzel, Alina Smorodchenko, Heidi Olze, Agnieszka J. Szczepek
{"title":"Tackling the Mouse-on-Mouse Problem in Cochlear Immunofluorescence: A Simple Double-Blocking Protocol for Immunofluorescent Labeling of Murine Cochlear Sections with Primary Mouse Antibodies","authors":"Mohamed Bassiouni,&nbsp;Katharina Stölzel,&nbsp;Alina Smorodchenko,&nbsp;Heidi Olze,&nbsp;Agnieszka J. Szczepek","doi":"10.1002/cpmo.84","DOIUrl":"10.1002/cpmo.84","url":null,"abstract":"<p>The mouse is the most widely used animal model in hearing research. Immunohistochemistry and immunofluorescent staining of murine cochlear sections have, thus, remained a backbone of inner ear research. Since many primary antibodies are raised in mouse, the problem of “mouse-on-mouse” background arises due to the interaction between the anti-mouse secondary antibody and the native mouse immunoglobulins. Here, we describe the pattern of mouse-on-mouse background fluorescence in sections of the postnatal mouse cochlea. Furthermore, we describe a simple double-blocking immunofluorescence protocol to label mouse cochlear cryosections. The protocol contains a conventional blocking step with serum, and an additional blocking step with a commercially available anti-mouse IgG blocking reagent. This blocking technique virtually eliminates the “mouse-on-mouse” background in murine cochlear sections, while adding only a little time to the staining protocol. We provide detailed instructions and practical tips for tissue harvesting, processing, and immunofluorescence-labeling. Further protocol modifications are described, to shorten the duration of the protocol, based on the primary antibody incubation temperature. Finally, we demonstrate examples of immunofluorescence staining performed using different incubation times and various incubation temperatures with a commercially available mouse monoclonal primary antibody. © 2020 The Authors.</p><p><b>Basic Protocol</b>: Tackling the Mouse-on-Mouse Problem in Cochlear Immunofluorescence: A Simple Double-Blocking Protocol for Immunofluorescent Labeling of Murine Cochlear Sections with Primary Mouse Antibodies</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.84","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38547018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment 小鼠眼组织和细胞外环境的定量蛋白质组学分析
Current protocols in mouse biology Pub Date : 2020-09-08 DOI: 10.1002/cpmo.83
Jason Yeung, Jeffrey Lamb, Jonathan R. Krieger, Mihaela Gadjeva, Jennifer Geddes-McAlister
{"title":"Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment","authors":"Jason Yeung,&nbsp;Jeffrey Lamb,&nbsp;Jonathan R. Krieger,&nbsp;Mihaela Gadjeva,&nbsp;Jennifer Geddes-McAlister","doi":"10.1002/cpmo.83","DOIUrl":"10.1002/cpmo.83","url":null,"abstract":"<p>Mass spectrometry-based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly <i>Pseudomonas aeruginosa</i>, and the formation of biofilms. The interplay between <i>P. aeruginosa</i> and the immune system at the site of infection defines the host's ability to defend against bacterial invasion and promote clearance of infection. Profiling of the ocular tissue following infection describes the nature of the host innate immune response and specifically the presence and abundance of neutrophil-associated proteins to neutralize the bacterial biofilm. Moreover, detection of unique proteins produced by <i>P. aeruginosa</i> enable identification of the bacterial species and may serve as a diagnostic approach in a clinical setting. Given the emergence and prevalence of antimicrobial resistant bacterial strains, the ability to rapidly diagnose a bacterial infection promoting quick and accurate treatment will reduce selective pressure towards resistance. Furthermore, the ability to define differences in the host immune response towards bacterial invasion enhances our understanding of innate immune system regulation at the ocular surface. Here, we describe murine ocular infection and sample collection, as well as outline protocols for protein extraction and mass spectrometry profiling from corneal tissue and extracellular environment (eye wash) samples. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Murine model of ocular infection</p><p><b>Basic Protocol 2</b>: Murine model sample collection</p><p><b>Basic Protocol 3</b>: Protein extraction from eye wash</p><p><b>Basic Protocol 4</b>: Protein extraction from corneal tissue</p><p><b>Basic Protocol 5</b>: Mass spectrometry-based proteomics and bioinformatics from eye wash and corneal tissue samples</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.83","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38353055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Assessment of Problem-Solving Skills and Inhibitory Control in Mice Using Water Escape Detour Test 水逃逸绕道实验对小鼠问题解决能力和抑制控制的评价
Current protocols in mouse biology Pub Date : 2020-09-01 DOI: 10.1002/cpmo.82
Grzegorz R. Juszczak, Anna Bobrowska
{"title":"Assessment of Problem-Solving Skills and Inhibitory Control in Mice Using Water Escape Detour Test","authors":"Grzegorz R. Juszczak,&nbsp;Anna Bobrowska","doi":"10.1002/cpmo.82","DOIUrl":"10.1002/cpmo.82","url":null,"abstract":"<p>Despite the importance of emotional intelligence, its biological mechanism is still not well understood. For this reason, we have developed a rodent detour task which requires an animal to reach a highly desired object placed directly behind a transparent barrier that blocks the direct route to the target. This apparently simple task is highly dependent on the emotional control that is necessary to inhibit prepotent and counterproductive responses driven by the sight of a desired object. The water escape detour task designed for mice enables testing the ability to solve emotionally challenging problems, as well as identification of an impairment termed perseveration. Such a maladaptive reaction to a challenging situation is characterized by difficulty in terminating an unsuccessful response, leading to persistent repetition of inappropriate behavior. This issue is important because perseveration is associated with schizophrenia, drug abuse, and aging. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Water escape detour task</p><p><b>Support Protocol 1</b>: Preparation of escape platform</p><p><b>Support Protocol 2</b>: Preparation of the transparent barrier</p><p><b>Alternate Protocol</b>: Water escape detour task for testing acute effects</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.82","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38331038","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Simultaneous Assessment of Circadian Rhythms and Sleep in Mice Using Passive Infrared Sensors: A User's Guide 使用被动红外传感器同时评估小鼠的昼夜节律和睡眠:用户指南
Current protocols in mouse biology Pub Date : 2020-08-31 DOI: 10.1002/cpmo.81
Laurence A. Brown, Gareth T. Banks, Neil Horner, Sian L. Wilcox, Patrick M. Nolan, Stuart N. Peirson
{"title":"Simultaneous Assessment of Circadian Rhythms and Sleep in Mice Using Passive Infrared Sensors: A User's Guide","authors":"Laurence A. Brown,&nbsp;Gareth T. Banks,&nbsp;Neil Horner,&nbsp;Sian L. Wilcox,&nbsp;Patrick M. Nolan,&nbsp;Stuart N. Peirson","doi":"10.1002/cpmo.81","DOIUrl":"10.1002/cpmo.81","url":null,"abstract":"<p>The 24-hr cycle of activity and sleep provides perhaps the most familiar example of circadian rhythms. In mammals, circadian activity rhythms are generated by a master biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). This clock is synchronized (entrained) to the external light environment via light input from retinal photoreceptors. However, sleep is not a simple circadian output and also is regulated by a homeostatic process whereby extended wakefulness increases the need for subsequent sleep. As such, the amount and distribution of sleep depends upon the interaction between both circadian and homeostatic processes. Moreover, the study of circadian activity and sleep is not confined only to these specialized fields. Sleep and circadian rhythm disruption is common in many conditions, ranging from neurological and metabolic disorders to aging. Such disruption is associated with a range of negative consequences including cognitive impairment and mood disorders, as well as immune and metabolic dysfunction. As circadian activity and sleep are hallmarks of normal healthy physiology, they also provide valuable welfare indicators. However, traditional methods for the monitoring of circadian rhythms and sleep in mice can require separate specialized resources as well as significant expertise. Here, we outline a low-cost, non-invasive, and open-source method for the simultaneous assessment of circadian activity and sleep in mice. This protocol describes both the assembly of the hardware used and the capture and analysis of data without the need for expertise in electronics or data processing. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Assembly of a PIR system for basic activity and sleep recordings</p><p><b>Alternate Protocol</b>: Data collection using Raspberry Pi</p><p><b>Support Protocol</b>: Circadian analysis using PIR sensors</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.81","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38326895","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
Phenotyping in Mice Using Continuous Home Cage Monitoring and Ultrasonic Vocalization Recordings 利用连续家养笼子监测和超声发声记录小鼠的表型
Current protocols in mouse biology Pub Date : 2020-08-19 DOI: 10.1002/cpmo.80
Liane Hobson, Rasneer S. Bains, Simon Greenaway, Sara Wells, Patrick M. Nolan
{"title":"Phenotyping in Mice Using Continuous Home Cage Monitoring and Ultrasonic Vocalization Recordings","authors":"Liane Hobson,&nbsp;Rasneer S. Bains,&nbsp;Simon Greenaway,&nbsp;Sara Wells,&nbsp;Patrick M. Nolan","doi":"10.1002/cpmo.80","DOIUrl":"10.1002/cpmo.80","url":null,"abstract":"<p>Over the last century, the study of mouse behavior has uncovered insights into brain molecular mechanisms while revealing potential causes of many neurological disorders. To this end, researchers have widely exploited the use of mutant strains, including those generated in mutagenesis screens and those produced using increasingly sophisticated genome engineering technologies. It is now relatively easy to access mouse models carrying alleles that faithfully recapitulate changes found in human patients or bearing variants of genes that provide data on those genes’ functions. Concurrent with these developments has been an appreciation of the limitations of some current testing platforms, especially those monitoring complex behaviors. Out-of-cage observational testing is useful in describing overt persistent phenotypes but risks missing sporadic or intermittent events. Furthermore, measuring the progression of a phenotype, potentially over many months, can be difficult while relying on assays that may be susceptible to changes in the testing environment. In recent years, there has also been increasing awareness that measurement of behaviors in isolation can be limiting, given that mice attempt to hide behavioral cues of vulnerability. To overcome these limitations, laboratory animal science is capitalizing on progress in data capture and processing expertise. Moreover, as additional recording modes become commonplace, ultrasonic vocalization recording is an appealing focus, as mice use vocalizations in various social contexts. Using video and audio technologies, we record the voluntary, unprovoked behaviors and vocalizations of mice in social groups. Adoption of these approaches is undoubtedly set to increase, as they capture the round-the-clock behavior of mouse strains. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Continuous recording of home cage activity using the Home Cage Analyzer (HCA) system</p><p><b>Support Protocol</b>: Subcutaneous insertion of a radio frequency identification microchip in the inguinal area</p><p><b>Basic Protocol 2</b>: Continuous recording of mouse ultrasonic vocalizations in the home cage.</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.80","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38288222","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 8
Behavioral Phenotyping for Down Syndrome in Mice 唐氏综合征小鼠行为表型研究
Current protocols in mouse biology Pub Date : 2020-08-11 DOI: 10.1002/cpmo.79
Randall J. Roper, Charles R. Goodlett, María Martínez de Lagrán, Mara Dierssen
{"title":"Behavioral Phenotyping for Down Syndrome in Mice","authors":"Randall J. Roper,&nbsp;Charles R. Goodlett,&nbsp;María Martínez de Lagrán,&nbsp;Mara Dierssen","doi":"10.1002/cpmo.79","DOIUrl":"10.1002/cpmo.79","url":null,"abstract":"<p>Down syndrome (DS) is the most frequent genetic cause of intellectual disability, characterized by alterations in different behavioral symptom domains: neurodevelopment, motor behavior, and cognition. As mouse models have the potential to generate data regarding the neurological basis for the specific behavioral profile of DS, and may indicate pharmacological treatments with the potential to affect their behavioral phenotype, it is important to be able to assess disease-relevant behavioral traits in animal models in order to provide biological plausibility to the potential findings. The field is at a juncture that requires assessments that may effectively translate the findings acquired in mouse models to humans with DS. In this article, behavioral tests are described that are relevant to the domains affected in DS. A neurodevelopmental behavioral screen, the balance beam test, and the Multivariate Concentric Square Field test to assess multiple behavioral phenotypes and locomotion are described, discussing the ways to merge these findings to more fully understand cognitive strengths and weaknesses in this population. New directions for approaches to cognitive assessment in mice and humans are discussed. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preweaning neurodevelopmental battery</p><p><b>Basic Protocol 2</b>: Balance beam</p><p><b>Basic Protocol 3</b>: Multivariate concentric square field test (MCSF)</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.79","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38260681","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 5
Quantification of Neutrophil Extracellular Traps Isolated From Mouse Tissues 小鼠组织中分离的中性粒细胞胞外陷阱的定量分析
Current protocols in mouse biology Pub Date : 2020-07-23 DOI: 10.1002/cpmo.78
Rebecca L. Buckland, Alicia S. Wilson, Anne Brüstle
{"title":"Quantification of Neutrophil Extracellular Traps Isolated From Mouse Tissues","authors":"Rebecca L. Buckland,&nbsp;Alicia S. Wilson,&nbsp;Anne Brüstle","doi":"10.1002/cpmo.78","DOIUrl":"10.1002/cpmo.78","url":null,"abstract":"<p>One of the most intriguing functions of neutrophils is the production of neutrophil extracellular traps (NETs), which are formed when neutrophils decondense their internal DNA and extrude it along with cytotoxic proteins in a web-like structure. This process allows neutrophils to trap and kill pathogens, and is also associated with multiple hematological and autoimmune conditions. Due to their rapid degradation, there are many challenges in accurately and specifically detecting and quantifying NETs. Microscopy is the gold standard for NET detection, but is not optimal for large-scale screening. Furthermore, methods relying on detection of free DNA or on flow cytometry–based examination of NET-associated markers can be nonspecific, time-consuming, and expensive. Here, we describe an innovative, quick, specific, and inexpensive conventional flow cytometry method for detecting neutrophils on the verge of forming NETs. These methods utilize pulse-shaped analysis (PulSA) to distinguish resting neutrophils from those with decondensed DNA, a prerequisite for NET formation. An increase in DNA-diffuse neutrophils is found in cell populations after exposure to NET-inducing stimuli, consistent with the DNA decondensation expected during neutrophil NET formation. These populations are only observed in granulocytes, validating the specificity of this method. We describe protocols optimized for neutrophils retrieved from mouse blood, spleen, and bone marrow. The relative speed and simplicity of the method described here makes it a useful tool for detecting NET formation in large-scale experiments. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol</b>: Detection of nuclear decondensation in neutrophils from stimulated murine bone marrow</p><p><b>Alternate Protocol 1</b>: Detection of nuclear decondensation in neutrophils from splenocytes</p><p><b>Alternate Protocol 2</b>: Detection of nuclear decondensation in neutrophils from blood</p><p><b>Support Protocol 1</b>: Cryopreservation and defrosting of samples</p><p><b>Support Protocol 2</b>: Paraformaldehyde fixation of samples</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-07-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.78","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38742685","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Whole Mount In Situ Hybridization of Mid-Gestation Mouse Embryos 中期妊娠小鼠胚胎全坐骨原位杂交
Current protocols in mouse biology Pub Date : 2020-06-15 DOI: 10.1002/cpmo.77
Annemiek Beverdam
{"title":"Whole Mount In Situ Hybridization of Mid-Gestation Mouse Embryos","authors":"Annemiek Beverdam","doi":"10.1002/cpmo.77","DOIUrl":"10.1002/cpmo.77","url":null,"abstract":"<p>In situ hybridization is a powerful technique that allows the visualization of specific RNA species in biological samples in exquisite detail. It has been particularly well explored in the field of developmental genetics. The spatial and temporal patterns of RNA expression provide us with critical information on likely gene function during embryonic development, and often inform the decision on whether to attempt further gene manipulation approaches. Furthermore, once a mouse strain with altered gene function has been created, in situ hybridization is a critical tool for revealing how the development of embryos with the mutation differs from that of wild-type embryos, and thus infer the function of the altered gene. Here, a well-tested protocol used to visualize RNA expression in whole-mount mid-gestation mouse embryos ranging from 8.5 to 14.5 days post-coitum (dpc) is described. © 2020 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: RNA probe synthesis</p><p><b>Alternate Protocol</b>: Preparation of DNA template by PCR</p><p><b>Basic Protocol 2</b>: Embryo dissection</p><p><b>Basic Protocol 3</b>: Whole mount in situ hybridization</p><p><b>Support Protocol</b>: Generation of embryo powder</p>","PeriodicalId":37980,"journal":{"name":"Current protocols in mouse biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmo.77","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38044824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
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