وتم تمويل هذه الدراسات من قبل ويلكوم ترست والمملكة المتحدة والتهاب السحايا. وقدمت HBMEC خط خلية KS الدكتور كيم. أجريت التصوير مبائر والمجهر الإلكتروني في مرفق Bioimaging ولفسون ، جامعة بريستول. نود أيضا أن نشكر السيد آلان Leard ، الدكتور مارك جيبسون (جامعة بريستول) ، والسيد آلان تيلي (PerkinElmer) لما قدموه من مساعدة والمشورة.

<ol>
	<li>Virji, M., Makepeace, K., Moxon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+mechanisms+of+interactions+of+Opc-expressing+meningococci+at+apical+and+basolateral+surfaces+of+human+endothelial+cells;+the+role+of+integrins+in+apical+interactions.">Distinct mechanisms of interactions of Opc-expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions.</a> Molecular Microbiology. 14, 173-184 (1994).</li><li>Virji, M., Makepeace, K., Peak, I. R., Ferguson, D. J., Jennings, M. P., Moxon, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opc-+and+pilus-+dependent+interactions+of+meningococci+with+human+endothelial+cells:+molecular+mechanisms+and+modulation+by+surface+polysaccharides.">Opc- and pilus- dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides.</a> Molecular Microbiology. 18, 741-754 (1995).</li><li>Sa E Cunha, C., Griffiths, N. J., Murillo, I., Virji, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+Opc+invasin+binds+to+the+cytoskeletal+protein+alpha-actinin.">Neisseria meningitidis Opc invasin binds to the cytoskeletal protein alpha-actinin.</a> Cellular Microbiology. 11, 384-405 (2009).</li><li>Virji, M., Kayhty, H., Ferguson, D. J., Alexandrescu, C., Alexandrescu, J. E., Heckels, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pili+in+the+interactions+of+pathogenic+Neisseria+with+cultured+human+endothelial+cells.">The role of pili in the interactions of pathogenic Neisseria with cultured human endothelial cells.</a> Molecular Microbiology. 5, 1831-1841 (1991).</li><li>Manders, E. M. M., Verbeek, F. J., Aten, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+colocalization+of+objects+in+dual+color+confocal+images.">Measurement of colocalization of objects in dual color confocal images.</a> Journal of Microscopy. 6, 357-382 (1993).</li><li>Zinchuk, V., Zinchuk, O., Okada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+colocalization+analysis+of+multicolor+confocal+immunofluorescence+microscopy+images:+pushing+pixels+to+explore+biological+phenomena.">Quantitative colocalization analysis of multicolor confocal immunofluorescence microscopy images: pushing pixels to explore biological phenomena.</a> Acta Histochemica et Cytochemica. 40, 101-111 (2007).</li></ol>

الإعلان عن أي تضارب في المصالح.

إمكانية ربط المنضوية OPC ، معربا عن النيسرية السحائية لα - actinin تم استكشافها من قبل باستخدام HBMEC فحص colocalisation من البكتيريا والبروتين هيكل الخلية في الخلايا المصابة بعد 3 و 8 فترة حضانة ح. بواسطة الفحص المجهري متحد البؤر ، يمكن أن يكون أظهر colocalisation من النيسرية السحائية مع α - actinin. على وجه الخصوص ، على الرغم من أن البكتيريا المنضوية في 3 ساعات ، كان هناك القليل مع colocalisation α - actinin في هذه النقطة الزمنية. وكانت أعداد كبيرة من الجراثيم البكتيرية الجمعيات مع هيكل الخلية البروتين يبدو بحاجة إلى فترة أطول من الإقامة داخل الخلايا وبعد فترة العدوى ح 8 ، α - actinin على ما يبدو في ارتباط وثيق. ألفا actinin هو بروتين متعددة الوظائف ، والتفاعلات البكتيرية مع هيكل الخلية يمكن أن يكون عنصر تأثير كبير على وظيفة خلايا الهدف الذي هو موضوع الدراسات الحالية. الكمي للcolocalisation على النحو المبين أعلاه يتطلب إعداد دقيق العينة. وينبغي إيلاء اهتمام خاص لتثبيت العينة ، وعرقلة الفترة والتخفيفات الأضداد. للحصول على أفضل إشارة إلى نسبة الضوضاء ، ينبغي معاير كل الأضداد الابتدائية والثانوية في التجارب الأولية لتحديد تركيزات الأمثل. في تجربتنا ، أنتجت Mowiol المتوسطة المتزايدة أفضل الصور.

1. الميكروسكوب الضوئي ليزر متحد البؤر (CLSM) : ايكا SP5 نظام التصوير مبائر : هذا النظام ، وذلك باستخدام مزيج من AOTF (صوتية البصرية تصفية tuneable) وAOBS ملف (صوتية بصرية شعاع التقسيم) ، ويبسط الإثارة مع أطوال موجة معينة. 2 - البرامج : ايكا مبائر LCS البرنامج ، لايكا مايكروسيستمز ، ألمانيا. Volocity 5 ، الارتجال ، PerkinElmer ، الولايات المتحدة الأمريكية.

visualisation and quantification of intracellular interactions of neisseria meningitidis and human &alpha;-actinin by confocal imaging

البروتين بمسانده من النيسرية السحائية (نيوتن متر ، المكورة السحائية) هي أرض لا يتجزأ من البروتين وأعربت الغشاء الخارجي ، والتي يمكن أن تكون بمثابة adhesin وفعالة لالإنفازين الإنسان الخلايا الظهارية والبطانية. حددنا البطانية ارض تقع لل integrins باسم المستقبلات الرئيسية لمكتب أمين المظالم ، وهي عملية تتطلب OPC لربط أول يغاندس إنتغرين مثل vitronectin وعبر هذه إلى خلايا مستقبلات أعرب 1. هذه العملية يؤدي إلى الغزو الجرثومي للخلايا بطانة الأوعية الدموية 2. في الآونة الأخيرة ، لاحظنا التفاعل مع بمسانده من البروتين الموجود في 100kDa lysates خلية كاملة من الخلايا البشرية 3. لاحظنا في البداية هذا التفاعل عندما بروتينات الخلية المضيفة مفصولة الكهربي ونشف إلى النيتروسليلوز كانوا مضافين مع OPC ، معربا عن نيوتن متر. كان التفاعل المباشر ، ولم يتضمن جزيئات متوسطة. بواسطة مطياف الكتلة ، أنشأنا هوية البروتين كما α - actinin. كما أعرب عن أي سطح عثر α - actinin على أي من خطوط الخلايا eight فحصها ، كما والتفاعلات OPC مع الخلايا البطانية في وجود الرصاص في الدم للدخول البكتيريا الى الخلايا المستهدفة ، ونحن دراسة إمكانية التفاعل البروتينين intracellularly. لهذا ، أصيب الاوعية الدموية الدقيقة في الدماغ البشري مثقف الخلايا البطانية (HBMECs) مع OPC ، معربا عن نيوتن متر لفترات طويلة والمواقع من البكتيريا والمنضوية α actinin ، تم فحص المجهري متحد البؤر التي كتبها. لاحظنا الوقت التي تعتمد على زيادة في colocalisation نيوتن متر من البروتين مع هيكل الخلية ، التي كانت كبيرة بعد فترة ثماني ساعات من تدخيل البكتيرية. بالإضافة إلى ذلك ، مكن استخدام برامج التصوير الكمي لنا للحصول على قياس نسبي لcolocalisation نيوتن متر مع α - actinin هيكل الخلية والبروتينات الأخرى. هنا نقدم بروتوكول للمرئيات والكمي لcolocalisation للبكتيريا مع البروتينات داخل الخلايا البكتيرية بعد دخوله إلى الخلايا البطانية الإنسان ، على الرغم من أن هذا الإجراء ينطبق أيضا على الخلايا الظهارية البشرية.

Watch this Scientific Journal Video about Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human Î±-actinin by Confocal Imaging at JoVE.com

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

يمكن النيسرية السحائية (نيوتن متر) ، وهي سلبية غرام الممرض التنفسي للإنسان على وجه التحديد ، ربط الإنسان actinin - α. هنا نقدم بروتوكول للتصور colocalisation للبكتيريا مع الخلايا α - actinin بعد دخول البكتيريا إلى خلايا الاوعية الدموية الدقيقة في الدماغ البطانية (HBMECs).

التخيل والكمي لالتفاعلات داخل الخلايا النيسرية السحائية والإنسان α - actinin بواسطة التصوير متحد البؤر

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

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Immunology and Infection

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

18.0K Views.  University of Bristol, UK. يمكن النيسرية السحائية (نيوتن متر) ، وهي سلبية غرام الممرض التنفسي للإنسان على وجه التحديد ، ربط الإنسان actinin - α. هنا نقدم بروتوكول للتصور colocalisation للبكتيريا مع الخلايا α - actinin بعد دخول البكتيريا إلى خلايا الاوعية الدموية الدقيقة في الدماغ البطانية (HBMECs).

Video: Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

القياسات خلية واحدة من تمزق رغوي الناجمة عن مسببات الأمراض بين الخلايا

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

التصوير InlC إفراز المعنية بالتحقيق في العدوى البكتيرية الخلوية من قبل الممرض<em&gt; المستوحدة الليستيريا</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Discovery of New Intracellular Pathogens by Amoebal Coculture and Amoebal Enrichment Approaches

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at all on selective media that are usually used to cultivate bacteria. For this reason, many of these pathogens were discovered only recently or following important outbreaks. These pathogens are often associated with amoebae, which serve as host-cell and allow the survival and growth of the bacteria. We intend here to provide a demonstration of two techniques that allow isolation and characterization of intracellular pathogens present in clinical or environmental samples: the amoebal coculture and the amoebal enrichment. Amoebal coculture allows recovery of intracellular bacteria by inoculating the investigated sample onto an amoebal lawn that can be infected and lysed by the intracellular bacteria present in the sample. Amoebal enrichment allows recovery of amoebae present in a clinical or environmental sample. This can lead to discovery of new amoebal species but also of new intracellular bacteria growing specifically in these amoebae. Together, these two techniques help to discover new intracellular bacteria able to grow in amoebae. Because of their ability to infect amoebae and resist phagocytosis, these intracellular bacteria might also escape phagocytosis by macrophages and thus, be pathogenic for higher eukaryotes.

Amoebal coculture is a cell culture system using adherent amoebae to selectively grow intracellular pathogens able to resist phagocytic cells such as amoebae and macrophages. It thus represents a key tool to discover new infectious agents. Amoebal enrichment allows discovery of new amoebal species and of their specific intracellular bacteria.

اكتشاف جديد بين الخلايا مسببات الأمراض عن طريق Amoebal Coculture والمناهج Amoebal التخصيب

Intracellular pathogens such as legionella, mycobacteria and Chlamydia-like organisms are difficult to isolate because they often grow poorly or not at ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

التصوير في الوقت الحقيقي من خلايا الدم النخاعي في ديناميات<em&gt; APC<sup&gt; مين / +</sup</em&gt; الأورام المعوية التي كتبها غزل القرص متحد البؤر المجهر

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

طريقة جديدة لتحليل نوعي متعدد حجم البكتيرية الأغشية الحيوية على شعيري الفطرية المستعمرات عن طريق متحد البؤر والمجهر الإلكتروني

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

تحقيقات الفحص البصري للرواية الخاصة بالبكتيريا في أنسجة الرئة البشرية السابقين فيفو بالليزر [كنفوكل] اندوميكروسكوبي

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

تصور داخل الخلايا المطوقة الاتجار بواسطة عمر Fluorescence التصوير المجهري

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

السابقين فيفو التهاب الغشاء المخاطي للأعضاء التناسلية أنثى والأنسجة اللمفاوية البشرية مع فيروس نقص المناعة البشرية 1 وهيستوكولتوري

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

التحديد الكمي افيروسيتوسيس بالفحص المجهري Fluorescence خلية واحدة

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

تصوير [كنفوكل] الحمض النووي الريبي مزدوج-الذين تقطعت بهم السبل ونمط الاعتراف مستقبلات في الإصابة بفيروسات الرنا الشعور السلبي

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...