وأيد هذا البحث من قبل شعبة البحوث ضمن الجدران من المعهد الوطني للحساسية والأمراض المعدية والمعاهد الوطنية للصحة. ونحن ممتنون لHuppa يوهانس ومارك ديفيس لتزويدنا scFv من H57 المستخدمة في هذه الدراسات. وأود أن أشكر RV كير نيومان للمناقشة مفيدة خلال تطوير هذه التقنية.

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تمت المحافظة على جميع الحيوانات المستخدمة في هذه التجارب في بيئة خالية من العوامل المسببة للأمراض محددة، وتمت الموافقة على هذه التجارب من قبل المعاهد الوطنية للصحة ورعاية الحيوان لجنة الاستخدام.

نحن هنا وصف نظام لدراسة الإشارات في مستضد محدد أساسي خلايا تي فأر باستخدام TIRFM وطبقات ثنائية زجاج الدهون كما دعمت ناقلات الجنود المدرعة الاصطناعي. هذا الاسلوب يعتمد على التعبير عن البروتينات بنجاح GFP المفتاحية في هذه الخلايا. Transfecting الخلايا التائية هي دائما مهمة صعبة. عادة، يتم استخدام electroporation أو الجينات تسليم باستخدام الفيروسات أو lentiviruses. أسلوب واحد ليست متفوقة على الآخر، وكلاهما يكون لها حدود ومزاياها. نجد أن electroporation والمزايا التالية: 1) لا يشترط أن يتم تقسيم خلايا نشطة كما هو مطلوب من أجل الفيروسات ولكن ليس lentiviruses، و 2) يمكن التحكم في مستوى التعبير من خلال تغيير وقت يتم تحضين الخلايا قبل التصوير. والعائق الاكبر هو أننا نجد صعوبة بالغة في التعبير عن البروتينات التي تكون كبيرة في الحجم في خلايا أولية. لقد قدمنا ​​الأسلوب الذي يعطينا جيد جدا بقاء الخلية بعد electroporation. ونتيجة لذلك فمن applicabجنيه إلى استخدامه لتحقيق إسكات الجينات بوساطة سيرنا. وصفنا أيضا هنا مخصصة 2 قناة TIRF اكتساب المجهر في وقت واحد الذي تم تصحيحه لونيا ولا يتطلب المواءمة منفصلة لأطوال موجية مختلفة. هذه القدرات، ومع ذلك، متوفرة تجاريا. ويستند نظامنا على مبادئ الفحص المجهري TIRF نشرت من قبل خبراء في الميدان، وأرخص من البدائل التجارية. واحد من انتقاد ممكن تصميم لدينا هو اننا نستخدم نفس الزاوية من حدوث موجات لإثارة مختلف. هذا من شأنه أن يؤدي إلى عمق تغلغل مختلف TIRF لإثارة موجات مختلفة. ونحن نعتقد أن هذه الآثار هي صغيرة لأن موجة زائل هو حقل تدهور بشكل كبير وعمق الميدان TIRF هي وظيفة خطية من الطول الموجي. وسوف Fluorophores على مقربة من سطح الزجاج تجربة لا فرق في شدة الليزر بين موجات اثنين. لfluorophores بالقرب من البولي ايثيلينعمق netration، فإن اختلاف كثافة يقدر بحوالي 1.3 أضعاف لموجات 2 488 و 640 نانومتر، وأضعاف 1.15 لموجات 2 488 و 561 نانومتر. ويمكن استخدام نفس النظام جنبا إلى جنب مع تقنيات فائقة أن قرار الاستفادة من الإضاءة TIRF. وقد وصفت نحن أيضا نظام كاميرا 2 التي بنيت العرف. مثل نظام TIRF، أجهزة مماثلة كما تتوفر تجاريا. نظامنا يوفر مرونة لتغيير الفلاتر وdichroics، مما يتيح استخدامه مع مجموعات مختلفة من الموجات. العيب في نظامنا هو درجة واحدة من دوران اللازمة لتحقيق المواءمة بين الصورتين. يمكن حل هذه المشكلة باستخدام كاميرا المدرجات التي يقودها بيزو وتقدم درجة التناوب المحاذاة. لدينا أيضا بنجاح تنفيذ نظام الكاميرا اثنين على هذا المجهر، الذي الكاميرات مختلفة ولها أحجام مختلفة بكسل. ومن شأن مثل هذا النظام أن يكون مفيدا إذا كان أحد يحتاج الأمر إلى حساسية في قناة واحدة ودى كبيرمجموعة namic في بلد آخر، وكلاهما نادرا ما تتوفر في نفس الكاميرا. استخدمنا HQ-2 Photometrics كاميرا في binning 2X2 تعطينا حجم بكسل فعالة من 12.9 ميكرون مع الكاميرا كوانت-EM Photometrics التي لديها حجم بكسل من 16 ميكرون. تم استخدام أنبوب 180 مم البعد البؤري للعدسة EM-كوانت وتم استخدام أنبوب 145 ملم طول بؤري عدسة للكاميرا HQ-2. تمت السيطرة على HQ-2 باستخدام Metamorph البرمجيات وتمت السيطرة كوانت-EM عبر الصغرى مدير البرنامج على جهاز كمبيوتر مستقل في وضع الزناد الخارجية. وقدمت على الزناد خارجي الى الكاميرا كوانت-EM باستخدام الإخراج الرقمي من لجنة المساعدة الإنمائية مجلس قياس والحوسبة، والذي تم تنفيذه باعتباره مصراع إضافية في إعدادات الإضاءة من تكوين Metamorph. نسبة أطوال البؤرية من العدسات أنبوب يطابق نسبة من أحجام بكسل فعالة. ويرد محاذاة ممثل في الشكل 5.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> Amaxa Nucleofector عدة لخلايا تي ماوس </td><td> Lonza المؤتمر الوطني العراقي. </td><td> VPA-1006 </td><td></td></tr><tr><td> PureLink HiPure البلازميد Midiprep كيت </td><td> الحياة تكنولوجيز </td><td> K2100-05 </td><td> لمحضرات الذيفان الداخلي الحمض النووي مجانا </td></tr><tr><td> Amaxa Nucleofector الأجهزة </td><td> Lonza المؤتمر الوطني العراقي. </td><td> AAD-1001 </td><td></td></tr><tr><td> المؤتلف الفئران IL-2 </td><td> Peprotech المؤتمر الوطني العراقي. </td><td> 212-12 </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent المجهرية 0.20μm </td><td> Polysciences، المؤتمر الوطني العراقي. </td><td> 24050-5 </td><td></td></tr><tr><td> مختبر تيك الثاني 8-جيدا حجرات ساترة </td><td> الحرارية العلمية، نونك </td><td> 155409 </td><td></td></tr></tbody></table>

a tirf microscopy technique for real-time, simultaneous imaging of the tcr and its associated signaling proteins

وبدأت إشارات من خلال مستقبلات الخلايا التائية (TCR) عندما تشارك بها شظايا الببتيد مستضدي ملزمة بمركب التوافق النسجي الأكبر (الشركة الفلسطينية للرهن) البروتينات وأعرب على سطح الخلايا مستضد تقديم (ناقلات الجنود المدرعة). ويتألف المجمع من TCR TCRαβ مثنوي مغاير يجند ملزم أن الزميلة غير تساهمي مع CD3 dimers (وεδ وεγ heterodimers وhomodimer ζζ) 1. على إشراك مستقبلات، وفسفرته سلاسل ζ CD3 من قبل عائلة كيناز SRC، LCK. هذا يؤدي إلى تجنيد من عائلة كيناز SYK، Zap70 الذي فسفرته ثم وتفعيلها من خلال LCK. بعد ذلك، Zap70 phosphorylates محول البروتينات اللات وSLP76، والشروع في تشكيل مجمع الإشارات القريبة التي تحتوي على عدد كبير من مختلف الجزيئات يشير الى 2. تشكيل هذه النتائج في نهاية المطاف في مجمع الكالسيوم وهيئة تنظيم الاتصالات التي تعتمد على رأسnscription تفعيل عامل والشروع يترتب على ذلك من سلسلة معقدة من البرامج التعبير الجيني التي تؤدي إلى تمايز الخلايا T 2. إشارات TCR (والدولة الناتجة من تمايز) هي عن طريق التضمين العديد من العوامل الأخرى، بما في ذلك قوة مستضد والحديث المتبادل مع co-stimulatory/co-inhibitory، chemokine، ومستقبلات خلوى 3-4. دراسة التنظيم المكاني والزماني للمجمع إشارات الداني في ظل ظروف تنشيط مختلف ولذلك فإن المفتاح لفهم المسار إشارات TCR، فضلا عن تنظيمها من قبل مما يشير الى مسارات أخرى. نظام واحد نموذجا مفيدا جدا لدراسة الإشارات التي بدأها TCR في غشاء البلازما في الخلايا التائية هي طبقات ثنائية الزجاج المدعومة من الدهون، كما هو موضح سابقا 5-6. ويمكن استخدام ما لتقديم مجمعات الشركة الفلسطينية للرهن المستضدية، التصاق، والجزيئات شارك في تنشيطية لخلايا T-بمثابة ناقلات الجنود المدرعة الاصطناعي. بواسطة التصوير خلايا تي التفاعل معطبقة ثنائية الدهن باستخدام مجموع مضان انعكاس داخلي المجهري (TIRFM)، يمكننا الحد من الإثارة على بعد 100 نيوتن متر من المسافة بين الزجاج وسطح الخلية 7-8. هذا يسمح لنا صورة الأحداث التي وقعت في المقام الأول يشير تحدث في غشاء البلازما. كما نحن مهتمون في مجال التصوير تجنيد البروتينات مما يشير إلى مجمع TCR، ونحن تصف اثنين من الكاميرا نظام التصوير TIRF فيه على TCR، وصفت مع فاب فلوري (مستضد جزء ملزم) أجزاء من الجسم المضاد H57 (تنقيته من ورم هجين H57-597 ، آي تي ​​سي سي، عدد ATCC: لا يجوز تصوير HB-218) التي هي محددة لTCRβ، والبروتينات مما يشير، الموسومة ب GFP، في وقت واحد في الوقت الحقيقي. هذه الاستراتيجية هو ضروري نظرا لطبيعة ديناميكية للغاية من خلايا تي وكلا من الأحداث يشير إلى أن تحدث في TCR. وقد سمحت هذه طريقة من طرق التصوير الباحثين ليغاندس صورة واحدة 9-11، فضلا عن تجنيد من الجزيئات مما يشير إلى تنشيط مستقبلات وهو نظام ممتاز لدراسة الكيمياء الحيوية في الموقع 12-16.

Watch this Scientific Journal Video about A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins at JoVE.com

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

والتقسيم من البروتينات، سواء داخل غشاء البلازما أو في مواقع داخل الخلايا هي واحدة الآلية التنظيمية التي يمكن أن تؤثر بشكل كبير نتائج إشارة، وبالتالي فهم إشارات من المهم دراسة السلوك المكاني والزماني للبروتينات المعنية. نحن هنا وصف نظام الفحص المجهري TIRF يستند إلى دراسة نقل الإشارات في الخلايا التائية، ولكن قابلة للتطبيق على نطاق واسع.

تقنية الفحص المجهري للTIRF التصوير، في الوقت الحقيقي في وقت واحد من TCR وما يرتبط بها من البروتينات اشارة

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

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Immunology and Infection

23.7K Views.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. والتقسيم من البروتينات، سواء داخل غشاء البلازما أو في مواقع داخل الخلايا هي واحدة الآلية التنظيمية التي يمكن أن تؤثر بشكل كبير نتائج إشارة، وبالتالي فهم إشارات من المهم دراسة السلوك المكاني والزماني للبروتينات المعنية. نحن هنا وصف نظام الفحص المجهري TIRF يستند إ...

Video: A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

في الوقت الحقيقي التصوير باء Leukotriene 4 الهجرة خلية وساطة BLT1 التفاعلات مع arrestin - β

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

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 ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

التصوير في الوقت الحقيقي من غشائي المفارق خلايا العدلات خلية أثناء التهجير تحت الفسيولوجية تدفق

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

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 ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

الكمي في وقت واحد T-خلية الدوائر مستقبلات الختان (TRECs) والدوائر K-حذف إعادة التركيب الختان (KRECs) من قبل في الوقت الحقيقي PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

تحليل<em&gt; يرسينيا القولون</em&gt; المستجيب إزفاء في الخلايا المضيفة عن طريق بيتا لاكتاماز المستجيب الإنشطار

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

التصوير العدلات وحيدات في مساريقي الأوردة التي كتبها حيوي داخلي المجهري على الفئران مخدرة في الوقت الحقيقي

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

تنقية الحمض النووي الفيروسي للتعرف على البروتينات الفيروسية والخلوية المرتبطة بها

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

التصوير في الوقت الحقيقي والتحديد الكمي للتنمية بيوفيلم الفطرية باستخدام نظام تدفق دائرية ذات مرحلتين

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using the analysis of cell membrane receptor clusters as a model, we present a detailed protocol for this image analysis task using Fiji (ImageJ) and Matlab routines to: 1) define regions of interest and design masks adapted to these regions; 2) track the particles in fluorescence microscopy videos; 3) analyze the diffusion and intensity characteristics of selected tracks. The quantitative analysis of the diffusion coefficients, types of motion, and cluster size obtained by fluorescence microscopy and image processing provides a valuable tool to objectively determine particle dynamics and the consequences of modifying environmental conditions. In this article we present detailed protocols for the analysis of these features. The method described here not only allows single-molecule tracking detection, but also automates the estimation of lateral diffusion parameters at the cell membrane, classifies the type of trajectory and allows complete analysis thus overcoming the difficulties in quantifying spot size over its entire trajectory at the cell membrane.

Here, we present a protocol for single particle tracking image analysis that allows quantitative evaluation of diffusion coefficients, types of motion and cluster sizes of single particles detected by fluorescence microscopy.

معالجة البروتوكول لتحليل نشر وحجم الكتلة من مستقبلات الغشاء بالفحص المجهري Fluorescence الصور

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...