نشكر سيلفي ليسيج وجميع أفراد مختبر لقراءة نقدية للبروتوكول. وأيد هذا العمل من خلال منحة (MOP-77545) من المعاهد الكندية لأبحاث الصحة (CIHR). ويدعم ناتالي Labrecque بواسطة منحة دراسية كبار FRSQ وميليسا ماتيو تلقى الكسندر غراهام بيل NSERC منحة دراسات عليا كندا.

<html> 1. إعداد قبل الجراحة عولجت فئران وفقا لمبادئ توجيهية المجلس الكندي للرعاية الحيوان. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Anesthetise الفئران عن طريق الحقن من الكيتامين / زيلازين (150/300 ملغ / كلغ، والملكية الفكرية) أو عن طريق استنشاق isoflurane (2٪، والأكسجين 1L). إذا كانت متوفرة، يجب استخدام isoflurane لأنها تسمح لتحسين السيطرة على الوقت التخدير وعمق. أيضا، ويدير الأكسجين في نفس الوقت الذي تدعم الوظائف الفسيولوجية للحيوان. وعلاوة على ذلك، يتم التخلص مباشرة isoflurane بواسطة الرئتين، وبالتالي لا يتطلب عملية التمثيل الغذائي كبدي أو كلوي. </li><li style=";text-align:right;direction:rtl"> حقن البوبرينورفين (0،05 حتي 0،1 مغ / كغ، SC) أو مسكن آخر. </li><li style=";text-align:right;direction:rtl"> تطبيق مرهم للعين فأر قبل الجراحة لتجنب جفاف العين (إذا تم استخدام isoflurane). </li><li style=";text-align:right;direction:rtl"> تحقق من أن الفأر هو نائم بواسطة معسر مخلب والمتداول ذيله. إذا الماوس لا يتفاعل، المضي قدما في عملية جراحية. </li></ol> 2. في جراحة الطبقة = &quot;jove_content&quot;&gt; ومن الممكن إزالة 4 من العقد اللمفاوية السطحية (LN) عن طريق الجراحة: في الاربية (2) والعضدية 2. عزلة LN أسهل في الفئران العجاف الشباب (6-8 أسابيع) منذ وجود الأنسجة الدهنية في الفئران الأكبر سنا يمكن أن تتداخل مع العزلة LN. ومع ذلك، يتم التغلب على هذا القيد مع الممارسة المتكررة. لراحة الفئران، ونحن حصاد بحد أقصى 2 LNS في وقت واحد، وعندما نقوم الوقت طبعا التجارب، ونحن حصاد LNS كل يوم الثاني على التوالي. أيضا، للوقاية من العدوى، يمكن إعطاء المضادات الحيوية لفأر، ولكن لم نفعل ذلك والجروح وشفاء ما يرام. يحتاج المرء أن يكون على علم بأن اعتمادا على التصميم التجريبي، واستخدام المضادات الحيوية يمكن أن تؤثر في النتيجة لاحظ (على سبيل المثال إذا تم قياس استجابة لنوع من البكتيريا). لاحظ أن يتم تعقيم كل الأدوات بعد كل استخدام، وجرى الابقاء على ظروف معقمة طوال الإجراءات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع الماوس على جانبها. </li><li style=";text-align:right;direction:rtl"> توطين في المنطقة حيث سيتم إجراء incisioن، اعتمادا على LN تريد الحصاد. (الشكل 1). </li><li style=";text-align:right;direction:rtl"> تطبق الكلورهيكسيدين، أو مطهر آخر، إلى تلك المنطقة. </li><li style=";text-align:right;direction:rtl"> جعل شق صغير مع مقص حاد (حوالي 5 ملم) إما على الجزء الخلفي من الرجل الاماميه أو فوق مخلب هند بالقرب من البطن (الشكل 1). </li><li style=";text-align:right;direction:rtl"> إزالة الشعر فضفاض كنت قد قطع مع الملقط. إذا كنت تفضل ذلك، قد حلق الماوس قبل إجراء شق. ومع ذلك، فإنه يطيل الداخلي ونجد أنه ليس من الضروري منذ ذلك الحين، كما هو موضح هنا، فإنه لا يحسن الشفاء أو نقصان العدوى، والتي هي نادرة جدا على حد سواء. </li><li style=";text-align:right;direction:rtl"> تمتد شق مع 2 ملقط من أجل رؤية LN. قد شق تصل الى 10 ملم. ويظهر LN رمادي أو أغمق من الدهون المحيطة بها. </li><li style=";text-align:right;direction:rtl"> قرصة فآسيا (الغشاء الرقيق الذي يغطي الدهون والأنسجة) على رأس LN مع واحد ملقط وسحب خفيفة من دون كسر الأنسجة المحيطة. </li><li style=";text-align:right;direction:rtl"> ضع ملقط 2 إلى أقصى حد ممكن underneath في LN. مع ملقط الأولى، كسر اللفافة وإزالة LN. إذا كنت قد نجحت في حصد LN، وينبغي أن وصمة عار من الدم تظهر في الموقع حيث كانت تقع في السابق LN. نلاحظ أن المصارف LN في حل متساوي التوتر. هذا اختبار بسيط يسمح لك للتحقق من صحة التي تم استخراجها 1 LN والأنسجة وليس الدهون. </li><li style=";text-align:right;direction:rtl"> تحقق من عدم وجود الشعر في الجرح. </li><li style=";text-align:right;direction:rtl"> إغلاق شق طريق الالتزام الأجزاء 2 من الجلد معا (داخل البشرة)، وتدبيس لهم مقطع. نستخدم بعض كليب ميشال مع ملقط تطبيق. تثبيت واحد أو 2 لقطات اعتمادا على حجم من شق. في معظم الأحيان، لقطات تسقط عندما النسيج يشفي. </li></ol> 3. بعد العملية الجراحية العناية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة بعض المواد الغذائية الرطبة في الجزء السفلي من القفص وبالتالي فإن الفئران يمكن أن تغذي بسهولة بعد الجراحة. </li><li style=";text-align:right;direction:rtl"> حوالي 6 إلى 8 ساعات بعد الجراحة، والتحقق من الفئران للتأكد من أنها لا تواجه الألم. مراقبة بعناية سلوكهم (الموقف، مشية، فيteraction مع متجانس ومظهر من الفراء). إذا كان المظهر العام للفئران والفقراء، وإدارة جرعة ثانية من البوبرينورفين. في اليوم التالي يجب أن الفئران استعادة سلوكهم الطبيعي وإلا الاستمرار في إدارة البوبرينورفين. وينبغي أن الفئران لم تصل إلى النهاية التي سبق تحديدها وقبولها من قبل مجلس رعاية الحيوان الخاصة بك. قد ترغب في الحصول على المشورة والمساعدة من جهاز فني رعاية الحيوان المربى اليابس. </li></ol> 4. العقدة اللمفية المناولة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في المختبر، للحصول على تعليق خلية واحدة، وفصل LN وفقا للإجراءات المعتادة. نضغط LN ضد الشرائح متجمد والحصول بصورة روتينية 1-2 × 10 6 خلية لكل LN. بدلا من ذلك، ونحن نستعد للLN لاستخراج الحمض النووي الريبي. </li><li style=";text-align:right;direction:rtl"> وصمة عار على الخلايا وفقا للبروتوكول الخاص المعتاد. يجب عليك الحصول المتوقع ملامح الخلوي LN تدفق. </li></ol> 5. ممثل النتائج مثال ص مبعثر إلى الأمام والجانبويظهر الكثير من أجل LNS تحصد من الماوس تخدير عن طريق الجراحة أو من الماوس ضحى (الشكل 2). في لمحة والنسب المئوية للخلايا اللمفاوية في بوابة الحية هي نفسها مما يدل على أن LN جراحة هو أسلوب ملائم للخلايا الحصاد. <img alt="الشكل 1" src="/files/ftp_upload/3444/3444fig1.jpg" /> الشكل 1. الأربي والعضدية توطين LN ​​في الماوس. يقع أ LN الأربية فقط فوق مخلب هند، قليلا نحو البطن. ب. يقع LN العضدية وراء الرجل الاماميه. <img alt="الشكل 2" src="/files/ftp_upload/3444/3444fig2.jpg" /> الرقم التدفق الخلوي 2. الملف من الخلايا الليمفاوية التي تم الحصول عليها بعد عملية جراحية LN. وتظهر ملامح مبعثر إلى الأمام والجانب لLNS تحصد من anesthetized الماوس عن طريق الجراحة (A) أو من الماوس ضحى (B). كانت تحصد LNS، فصلها وتحليلها على تدفق عداد الكريات. ويبين النسب المئوية للخلايا اللمفاوية في بوابة الحي.

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الإعلان عن أي تضارب في المصالح.

وقد وصفت نحن بروتوكولا للجراحة LN التي يمكن أن تنطبق على كثير من النظم التجريبية. وعلى الرغم من المفيد جدا لمتابعة الاستجابات المناعية أو المناعي توازن الخلية، هذه التقنية لديها عدد قليل من القيود. أولا، يمكن أن يحصد سوى أربعة LNS. ثانيا، يجب أن يكون رد الفعل المناعي در?...

<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> غريف الجميلة الملقط بريميوم نمط </td><td> هارفارد جهاز </td><td> 52-2144 </td><td> منحني بقوة، 10 سم (4 بوصة)، و 0.8 ملم معلومات سرية </td></tr><tr><td> ميشال كليب تطبيق وإزالة الملقط </td><td> هارفارد جهاز </td><td> 52-3779 </td><td> 12.5 سم (5 بوصة) </td></tr><tr><td> ميشال كليب 100 </td><td> هارفارد جهاز </td><td> 52-3746 </td><td> 7،5-1،75 مم </td></tr><tr><td> Temgesic (البوبرينورفين هيدروكلوريد) </td><td> شيرينغ بلاو </td><td></td><td></td></tr><tr><td> Vetalar (هيدروكلوريد الكيتامين) </td><td> Boniche الحيوان للرعاية الصحية </td><td></td><td> الدين: 01989529 </td></tr><tr><td> Rompun (زيلازين) </td><td> باير </td> &lt;TD&gt; </td><td> الدين: 02169592 </td></tr><tr><td> Isoflurane </td><td> ABBOTT </td><td></td><td> الدين: 02032384 </td></tr><tr><td> Hypotears العين مرهم </td><td> نوفارتيس Ophtalmics </td><td></td><td> الدين: 02133288 </td></tr><tr><td> Baxedin (Clorhexidine جلوكونات 2٪ في ايزوبروبيل 70٪) </td><td> أوميغا </td><td> L0000017 </td><td> الدين: 02251477 </td></tr></tbody></table>

murine superficial lymph node surgery

في مجال علم المناعة، لفهم تطور استجابة مناعية ضد لقاح لعدوى أو ورم، ويتبع في كثير من الأحيان استجابة مع مرور الوقت. وبالمثل، فإن دراسة التوازن لمفاوية يتطلب بطبيعة الحال التجارب المرة. تنفيذ هذه الدراسات في الماوس نفسه مثالية للحد من التقلبات التجريبية، فضلا عن عدد من الفئران المستخدمة. سحب الدم يسمح أداء التجارب مسار الزمن، لكنه يعطي فقط معلومات حول تعميم الخلايا اللمفية، ويقدم لعدد محدود من الخلايا 1-4. منذ الخلايا الليمفاوية من خلال تعميم الجسد والمقيمين في الغدد الليمفاوية لها خصائص مختلفة، من المهم أن تدرس كلا الموقعين. إزالة متتابعة من الغدد الليمفاوية عن طريق الجراحة يوفر فرصة فريدة لمتابعة استجابة مناعية أو توسيع الخلية المناعية في الماوس نفسه مع مرور الوقت. وعلاوة على ذلك، هذا الأسلوب ينتج بين الخلايا 6 1-2x10 في العقدة الليمفاوية التي هي كافية لPErform التوصيف المظهري و / أو فحوصات وظيفية. وقد متتابعة لعملية جراحية العقدة الليمفاوية أو الغدد الليمفاوية استخدمت بنجاح من قبلنا وغيرها من 5-11. هنا، نحن تصف كيف يمكن إزالة العقد اللمفية العضدية، والاربية من خلال جعل شق صغير في الجلد من فأر anesthetised. منذ العملية الجراحية هو سطحي والقيام بسرعة، والماوس للشفاء بسرعة كبيرة، ويشفى تماما وليس تجربة الألم المفرط. كل يوم الثاني على التوالي، فمن الممكن لجني الغدد الليمفاوية واحد أو اثنين مما يسمح للتجارب دوام. هذا الأسلوب هو مناسب وبالتالي إلى دراسة خصائص العقدة الليمفاوية، الخلايا الليمفاوية المقيمين على مر الزمن. هذا النهج هو مناسبة لتصاميم تجريبية مختلفة، ونعتقد أن العديد من المختبرات ستستفيد من تنفيذ عمليات جراحية متتابعة العقدة الليمفاوية.

Watch this Scientific Journal Video about Murine Superficial Lymph Node Surgery at JoVE.com

Murine Superficial Lymph Node Surgery

لمتابعة تطور استجابة مناعية أكثر من مرة داخل الماوس نفسه، ويمكن إزالة الغدد الليمفاوية بشكل متتابع عن طريق الجراحة. هنا، نحن تصف كيف يمكن تنفيذ هذه التقنية.

الفئران عقدة اللمفاوية السطحية جراحة

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Research

JoVE Journal

Immunology and Infection

41.9K Views.  Maisonneuve-Rosemont Hospital Research Center. لمتابعة تطور استجابة مناعية أكثر من مرة داخل الماوس نفسه، ويمكن إزالة الغدد الليمفاوية بشكل متتابع عن طريق الجراحة. هنا، نحن تصف كيف يمكن تنفيذ هذه التقنية.

Video: Murine Superficial Lymph Node Surgery

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

Intravital الميكروسكوب من العقدة الليمفاوية الأربي

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

التصوير خارج الحي من خلايا تي في الفئران شرائح العقدة الليمفاوية مع Widefield ومجاهر متحد البؤر

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Intravital التصوير من العقدة اللمفية الفأر المأبضية

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

جيل من العقدة الليمفاوية الدسم سادة الوهم لدراسة العقدة الليمفاوية اللحمية خلية المنشأ

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

عزل خلايا الفئران العقدة الليمفاوية اللحمية

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

النخاعي عزل الخلايا من الجلد ماوس وتصريف العقدة الليمفاوية بعد الأدمة التحصين مع لايف الموهن<em&gt; المتصورة</em&gt; Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

الفئران كامل سماكة الجلد زراعة

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our group have shown that hypertension is associated with an excessive accumulation of T cells in the vessels and kidney during the development of experimental hypertension6. Once in these tissues, T cells produce several cytokines that affect both vascular and renal function leading to vasoconstriction and sodium and water retention1,2. To fully understand how T cells cause cardiovascular and renal diseases, it is important to be able to identify and quantify the specific T cell subsets present in these tissues. T cell subsets are defined by a combination of surface markers, the cytokines they secrete, and the transcription factors they express. The complexity of the T cell population makes flow cytometry and intracellular staining an invaluable technique to dissect the phenotypes of the lymphocytes present in tissues. Here, we provide a detailed protocol to identify the surface and intracellular markers (cytokines and transcription factors) in T cells isolated from murine kidney, aorta and aortic draining lymph nodes in a model of angiotensin II induced hypertension. The following steps are described in detail: isolation of the tissues, generation of the single cell suspensions, ex vivo stimulation, fixation, permeabilization and staining. In addition, several fundamental principles of flow cytometric analyses including choosing the proper controls and appropriate gating strategies are discussed.

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

بين الخلايا تلطيخ والتدفق الخلوي لتحديد الخلايا الليمفاوية مجموعات فرعية ضمن الفئران الأورطي، الكلى والغدد الليمفاوية في نموذج من ارتفاع ضغط الدم

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

جمع وتجهيز العقد الليمفاوية من الحيوانات الكبيرة لتحليل الحمض النووي الريبي: التحضير للدراسات ترانسكريبتوميك العقدة الليمفاوية للأنواع الحيوانية الكبيرة

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

تحديد المجموعات الفرعية التنظيمية تي الخلية في الغدة الصعترية مورين، البنكرياس تصب العقدة الليمفاوية والطحال باستخدام التدفق الخلوي

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...