معتمدة LWE وRK من قبل المعاهد الوطنية الأميركية للصحة منح R37 NS033506-16 و R01 NS060699-03. كان مدعوما من قبل المجتمع MPT لسرطان زمالة أبحاث ما بعد الدكتوراه الأمريكية (PF-10-057-01-MPC). كانت المشورة والمعرفة من الدكتور ماثيو ليمان والدكتور أورين Kobiler دور أساسي في تصميم التكوين المجهر وأساليب التصوير الخلية الحية. كما نتقدم بالشكر لنيل بارلو وبرايان كين T. من نيكون الأجهزة للمساعدة التقنية في اقتناء وتركيب وأداء المجهر.

<ol>
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ليس لدينا شيء في الكشف عنها.

الهدف من تصوير الخلايا الحية ومراقبة العمليات البيولوجية لأنها تحدث دون تغيير كبير بفعل المراقبة. ويتم تحقيق هذا الهدف عن طريق الاستفادة المثلى ثلاثة متغيرات: مراقبة البيئة، وسرعة التصوير، والإضاءة مضان. يجب أن يكون متوازنا بين هذه المتغيرات التي تعتمد لتحقيق ظروف ...

إصابة الجهاز العصبي المحيطي (السندات الإذنية) عن طريق الفيروسات الهربسي الألفائي مثل فيروس الهربس البسيط (HSV) -1، -2، وفيروس داء الكلب الكاذب (PRV) ينطوي على عدة خطوات معقدة ودرجة عالية من التنظيم في جميع مراحل الحياة الفيروسية. النقل داخل الخلايا العصبية في الجهاز العصبي المحيطي أمر بالغ الأهمية خلال أحداث كلا العدوى الفيروسية الأولية واللاحقة انتشار بين المضيف. الآليات الجزيئية التي تعدل عنصرين من دورة الحياة الفيروسية؛ النقل الموجهة من الجمعيات الفيروسية بعيدا عن أجسام الخلايا ضمن محاور عصبية (النقل تقدمي) ونقل لاحقا إلى خلايا virions عرضة (انتشار تقدمي) مهمة لفهم المرضية هربس. النقل والخروج من الجسيمات الفيروسية في الخلايا العصبية تعتمد على تجميع ناضجة المعدية 1،2 الفيريون. واستخدمت المقايسات ثابت سابقا، بما في ذلك المناعي (IF) والمجهر الإلكتروني (EM)، لدراسة حالة جمعية الجسيمات والبروتوكول الاضافيالتفاعلات عين المرتبطة بالنقل الفيريون وانتشار 3-6. إلا أن الطبيعة الديناميكية لنقل virions والتحف التجريبية التي أدخلتها التثبيت الكيميائية مرتبك تفسير الصور الثابتة 7،8. مؤخرا، قد تم وصفها عدد من البروتينات الانصهار الفيروسي الفلورسنت لفيروس الهربس البسيط وPRV التي لها تأثيرات ضئيلة على وظيفة البروتين. وغالبا ما يقترن بروتين الفلورية الخضراء (GFP) والبروتينات الفلورية الأحمر (mCherry أو mRFP) fluorophores للسماح التصوير لاثنين من المكونات الهيكلية ثلاثة من الفيريون ناضجة: قفيصة، غشاء، وبروتين سكري 9-11. يعيش التصوير الخلية النقل تقدمي استخدام مزدوج المسمى الفيروسات يتصور الدولة تجميع الجسيمات الفيروسية أثناء النقل. وتستخدم fluorophore مماثلة معربا عن سلالات فيروسية لتصور عدد وتنوع الجينوم الفيروسي بعد انتشار 12،13. خصائص البروتينات الفلورية (مراجعة في 14،15) تؤثر بشكل كبيرالقدرة على تصور جمعيات الفيروسية أو الخلوية. ينبغي النظر في الخصائص الذاتية للبروتين فلوري، بما في ذلك الذات التفاعلات والاستقرار، عند تصميم واختبار اندماج بروتين رواية للحفاظ على النوع البري وظيفة. بالتزامن مع اندماج بروتين فلوري، وهما جيدا تتميز في أنظمة زراعة الخلايا في المختبر وتستخدم لتصوير الخلايا الحية من عدوى فيروس الهربس: فصل 16 و 17 مجزأة (الشكل 1A) الفئران متفوقة عنق الرحم العقد (SCG) الخلايا العصبية. في كلا النظامين، يتم تشريح الجنينية SCG، وفصلها إلى هيئات خلية واحدة، ومطلي لفي المختبر زراعة 18. الخلايا العصبية SCG هي جزء من الجهاز العصبي اللاإرادي ويمكن تربيتها بسهولة ومتباينة من قبل عامل نمو الخلايا العصبية (NGF) في ناضجة الاستقطاب فيفو السابقين الدولة. الخلايا العصبية SCG فصلها تشكل شبكة ممتدة من المحاور التي يسمح جمعةأو التصور من الجسيمات الفيروسية لأنها تمر بعملية انتقال تقدمي 6. الثقافات SCG مجزأة توفير العزلة فلويديك من جسم الخلية العصبية (S مقصورة) ومصطلحات محوار البعيدة (N مقصورة) 19. وقد تم نشر عدد من بروتوكولات وإجراءات تفصيلية لبناء واستخدام الخلايا العصبية باستخدام مجزأة الأصلي أو تعديل ل20 Campenot الغرف 17 سابقا. العزلة فلويديك يسمح للعدوى انتقائية من أجسام الخلايا العصبية والكشف عن virions ذرية بعد النقل إلى مصطلحات محوار معزولة. الطلاء الخلايا الظهارية على مصطلحات مسبق للإصابة توفر الخلايا المتلقية لانتشار عدوى فيروسية. هناك العديد من العناصر الأساسية الهامة لجميع الخلايا الحية التجريب التصوير، ولكن الأكثر ملاءمة لبروتوكولات لدينا هي: اقتناء الآلي صورة، والإضاءة الفلورسنت، وسرعة التصوير والمراقبة البيئية. لجميع تجارب التصوير، ونحن نستخدممقلوب، الآلي، إضاءة المجهر epifluorescence (الشكل 1B). بنيت المجهر حول نيكون الكسوف تي قاعدة، وتوظف عددا من سيطرة النظم الآلية الكمبيوتر بسرعة إعادة تكوين المجهر خلال الحصول على الصور الآلي. لإضاءة مضان، ونحن نستخدم واسعة الطيف الزئبق مصباح القوس التي يمكن أن تكون مخففة مع مرشحات الكثافة محايدة على طول مسار إضاءة. مرشحات الإثارة لحد من الطيف من الإضاءة الفلورسنت وعندما يقترن مع المرايا مزدوج اللون متعددة تمرير والمرشحات الانبعاثات مضان تصور مركبات الفلورسنت محددة. هي التي شنت على مرشحات الإثارة وانبعاث في التحول بسرعة عجلات مستقلة، مرشح لتمكين اكتساب متتابعة السريع للقنوات الفلورسنت مختلفة. ومما يعزز من سرعة الحصول على الصور كذلك مع كاميرا EM-CCD حساسة وسريعة ومفيدة للكشف عن إشارات كثافة منخفضة وقصيرة صورة قراءة مرات. الرقابة البيئية على microscويتحقق مكتب مستشار رئيس الوزراء مع كبار مرحلة ساخنة الحاضنة وسخان عدسة الهدف للحفاظ على العينات عند درجات حرارة مرتفعة في حين يتم تمرير 2 المخصب جو مرطب وأول أكسيد الكربون في الحاضنة. يتم الاحتفاظ المجهر في غرفة مظلمة مع الحفاظ على درجة الحرارة المحيطة يقرب من 25 درجة مئوية، وضوء خارج الحد الأدنى مع ستائر التعتيم على جميع النوافذ. البروتوكولات التالية تصف استخدام هذا النظام لنقل تقدمي وانتشار المقايسات. وهناك عدد من البدائل موجودة للسيطرة على متغيرات التصوير الخلية الحية. السيطرة على إعدادات المجهري يمكن القيام بها يدويا أو من خلال النظم الآلية تعتمد على البرمجيات الاحتكارية. ويمكن تحقيق إضاءة الهالوجين مع مضان، LED أو مصادر الليزر. يتم تعديل سرعة التصوير من سرعة تبديل فلتر والوقت لتصور إشارة مع نظام الكشف المقترنة. ويمكن تحقيق الرقابة البيئية مع الأجهزة المتخصصة على ميلالمرحلة croscope، الضميمة من كل أو جزء من المجهر في مربع ساخنة ومرطب، أو عن طريق رفع درجة حرارة الغرفة حيث سيتم تنفيذ المجهري. كل من هذه البدائل لديها مزايا وعيوب تتعلق التكلفة والأداء. في بروتوكول لاحقة، ونحن من التفصيل استخدام التصوير الخلية الحية لدراسة النقل تقدمي السريع وانتشار تقدمي من الفيروسات الهربسي الألفائي من خلال استخدام سلالات فيروسية المؤتلف. في الوقت الحقيقي التصوير الخلية الحية يتصور قفيصة، غشاء، و / أو بروتين سكري شارك في التعريب على الهياكل الحيوية يمر النقل داخل محاور 11. التصوير تيميلابسي بين عشية وضحاها من الثقافات العصبية مجزأة يتصور محور عصبي الخروج الفيريون والعدوى من الخلايا عرضة 12. وقد تم تحسين البروتوكولات المعروضة هنا للاستخدام مع نظام التصوير خاصة لدينا، ولكن يتم عرض بعبارات عامة بالنسبة إلى العناصر الأربعة للتصوير الخلايا الحية. في المناقشة نحنسوف مزيد من التفاصيل بعض من التحسين ما هو ضروري لإجراء التجارب الناجحة.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>35 mm glass bottom culture dish</td>
 <td>MatTek Corporation; Ashland, MA </td>
 <td>P35G-1.5-20-C</td>
 </tr>
 <tr>
 <td>35 mm &mu;-Dish </td>
 <td>Ibidi USA LLC, Verona, WI </td>
 <td>81156</td>
 </tr>
 <tr>
 <td>Microscope body </td>
 <td>Nikon Instruments Inc. </td>
 <td>Nikon Eclipse Ti</td>
 </tr>
 <tr>
 <td>Motorized microscope X-Y stage</td>
 <td>Prior Scientific; Rockland, MA </td>
 <td>H117 ProScan Flat Top</td>
 </tr>
 <tr>
 <td>Motorized filter wheels</td>
 <td>Prior Scientific</td>
 <td>HF110 10 position filter wheel</td>
 </tr>
 <tr>
 <td>Fluorescent illumination source </td>
 <td>Lumen Dynamics; Mississauga, Ontario Canada </td>
 <td>X-Cite 120Q </td>
 </tr>
 <tr>
 <td>Multi-band Fluorescence filter sets</td>
 <td>Chroma Technology Corp.; Bellows Falls, VT</td>
 <td>89000 Sedat Quad - ET 89006 ECFP/EYFP/mCherry - ET</td>
 </tr>
 <tr>
 <td>EM-CCD camera</td>
 <td>Andor Technology USA; South Windsor, CT</td>
 <td>iXon3 897 </td>
 </tr>
 <tr>
 <td>Chamlide stage top environmental incubator</td>
 <td>Live Cell Instruments; Seoul, South Korea</td>
 <td>TC-L-10</td>
 </tr>
 <tr>
 <td>Objective lens heater</td>
 <td>Bioptecs; Butler, PA</td>
 <td>150803 Controller 150819-12-08 Heater</td>
 </tr>
 <tr>
 <td>Analysis software</td>
 <td>Nikon Instruments Inc.; Melville, NY</td>
 <td>NIS Elements </td>
 </tr>
 </tbody>
</table>

live cell imaging of alphaherpes virus anterograde transport and spread

وقد مكنت التطورات في تقنيات الخلية الحية المجهري مضان، فضلا عن بناء السلالات الفيروسية المؤتلف التي تعبر عن البروتينات الانصهار فلوري التصور في الوقت الحقيقي النقل وانتشار عدوى الفيروس الهربسي الألفائي من الخلايا العصبية. الأداة المساعدة من رواية البروتينات الانصهار الفلورسنت لغشاء الفيروسية، غشاء، وcapsids ألف، بالاشتراك مع التصوير الخلية الحية، حدد جمعيات الجسيمات الفيروسية تمر النقل داخل محاور عصبية. وقد استخدمت أدوات مشابهة بنجاح لتحليل خلية خلية انتشار الجسيمات الفيروسية لتحديد عدد وتنوع virions تنتقل بين الخلايا. الأهم من ذلك أن تقنيات تصوير الخلايا الحية النقل تقدمي وانتشار إنتاج ثروة من المعلومات بما في ذلك السرعات الجسيمات النقل والتوزيع من الجزيئات، والتحليلات الزمنية للتوطين البروتين. جنبا إلى جنب مع التقنيات الوراثية الفيروسية الكلاسيكية، وقد وفرت هذه المنهجيات رؤى الحرجةإلى أسئلة الآلية الهامة. في هذه المادة ونحن تصف بالتفصيل أساليب التصوير التي تم وضعها للإجابة على الأسئلة الأساسية للالهربسي الألفائي نقل الفيروس وانتشاره.

النقل تقدمي تطبيق هذا البروتوكول إلى التهابات الثقافات SCG فصلها مع PRV 348، سلالة PRV المؤتلف معربا عن GFP-US9 وGM-mCherry البروتينات الانصهار الغشاء، يسرت التصور من النقل تقدمي من virions (الشكل 3 و 4 فيلم التكميلي). إدراج هذه الب...

Watch this Scientific Journal Video about Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread at JoVE.com

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

تصوير الخلايا الحية من العدوى بالفيروس الهربسي الألفائي تمكن من تحليل للأحداث دينامية النقل الموجهة وانتشار بين الخلايا. هنا، فإننا نقدم المنهجيات التي تستخدم سلالات فيروسية المؤتلف معربا عن البروتينات الانصهار الفلورسنت لتسهيل التصور من الجمعيات الفيروسية خلال العدوى من الخلايا العصبية الأولية.

تصوير الخلايا الحية من الهربسي الألفائي الفيروسات النقل تقدمي وانتشارها

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

live-cell-imaging-alphaherpes-virus-anterograde-transport

Research

JoVE Journal

Immunology and Infection

11.0K Views.  Montana State University. تصوير الخلايا الحية من العدوى بالفيروس الهربسي الألفائي تمكن من تحليل للأحداث دينامية النقل الموجهة وانتشار بين الخلايا. هنا، فإننا نقدم المنهجيات التي تستخدم سلالات فيروسية المؤتلف معربا عن البروتينات الانصهار الفلورسنت لتسهيل التصور من الجمعيات الفيروسية ...

Video: Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

استخدام اعتراض البصرية لدراسة التفاعلات المرضي المضيف الحيوي للخلية الحية التصوير

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

تعيش خلية من التصوير<emالعصوية الرقيقة> و العقدية الرئوية الآلي باستخدام الميكروسكوب مرور الزمن

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

في الوقت الحقيقي تصوير لايف تي خلية اشارة تشكيل مجمع

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

عزل وتحليل العدلة لتحديد ما دورها في الغدد اللمفاوية حساسية الخلية وكلاء العلاجية

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

تقييم فعالية وسمية الرنا استهداف HIV-1 الإنتاج للاستخدام في الجينات أو العلاج بالعقاقير

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

تصوير تفاعل الخلية في الغشاء المخاطي الرغامى أثناء عدوى فيروس الإنفلونزا باستخدام الفحص المجهري إينترافيتال اثنين-فوتون

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

في اختبار فيترو ELISA لتقييم قوة لقاح داء الكلب

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

فيروس أنفلونزا مراسل لوسيفيراز الفلورسنت للتصوير الحي والقياس الكمي للعدوى الفيروسية

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) and other respiratory pathologies in vivo is difficult due to the limited accessibility and inherent motion artifacts of the lungs. Nonetheless, various approaches have been developed to overcome these challenges. This protocol describes a method for intravital fluorescence microscopy to study real-time leukocyte-endothelial interactions in the pulmonary microcirculation in an experimental model of ALI. An in vivo lung imaging system and 3-D printed intravital microscopy platform are used to secure the anesthetized mouse and stabilize the lung while minimizing confounding lung injury. Following preparation, widefield fluorescence microscopy is used to study leukocyte adhesion, leukocyte rolling, and capillary function. While the protocol presented here focuses on imaging in an acute model of inflammatory lung disease, it may also be adapted to study other pathological and physiological processes in the lung.

Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system.

الفحص المجهري الفلوري واسع المجال داخل الحيوية لدوران الأوعية الدقيقة الرئوية في إصابات الرئة الحادة التجريبية باستخدام نظام تصوير مستقر بالفراغ

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

التصوير المباشر في الوقت الحقيقي لتجديد الغدة تحت الفك السفلي للفأر

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

تسليط الضوء على المؤلف: دراسة تفاعلات الخلايا الظهارية البلاعم في تجديد الغدة اللعابية بعد الإصابة 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

استجواب تفاعلات الخلايا الخلوية في الغدة اللعابية عبر تصوير الخلايا الحية خارج الجسم الحي

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...