The authors thank Sanjiv Luther and colleagues for helpful discussions in establishing the current lymph node digestion protocol. This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R.

في هذا المنشور الفيديو والبروتوكول، وأجريت كافة الإجراءات الحيوانية وفقا لبروتوكول الحيوانية التي وافقت عليها السلطة كانتون بازل شتات، سويسرا. 1. إعداد العقد الليمفاوية والهضم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الماء قبل الحرارة في كوب إلى 37 درجة مئوية على النمام المغناطيسي مع لوحة التدفئة. </li><li style=";text-align:right;direction:rtl"> إعداد المتوسطة الأساسية على النحو التالي: DMEM المتوسطة (دون البيروفات) تستكمل مع FCS 2٪، 1.2 مم CaCl 2 و القلم / بكتيريا (100 وحدة من البنسلين، 100 ميكروغرام من الستربتومايسين). </li><li style=";text-align:right;direction:rtl"> تعقيم جميع الأدوات تشريح قبل الاستخدام. </li><li style=";text-align:right;direction:rtl"> الموت ببطء الفئران العقدة الليمفاوية المانحة في CO 2 الخنق وجو معقم و مطهر تشريح الغدد الليمفاوية. لا تشريح الدهون المحيطة بها. ملاحظة: تم تحسين هذا البروتوكول للالطرفية عقدة تجفيف الجلد الليمفاوية (الاربية، العضدية، الإبطين). </li><li style=";text-align:right;direction:rtl"> وضع الغدد الليمفاوية في طبق بتري معقمة تحتوي على 2 مل الجليد المتوسطة الأساسي البارد. </li><li style=";text-align:right;direction:rtl"> تعطيل كاليفورنيا العقدة الليمفاويةpsule باستخدام اثنين من الإبر 25 G ثابتة على 1 مل حقنة. </li><li style=";text-align:right;direction:rtl"> نقل الأنسجة العقدة الليمفاوية تعطلت في 5 مل البولي بروبلين أنبوب الجولة القاع تحتوي على 750 ميكرولتر المتوسطة الأساسي تستكمل مع 1 ملغ / مل كولاجيناز الرابع و 40 ميكروغرام / مل الدناز I. </li><li style=";text-align:right;direction:rtl"> إضافة واحد النمام المغناطيسي معقمة في كل أنبوب. </li><li style=";text-align:right;direction:rtl"> مكان أنبوب في الدورق مع 37 ° C مسخن الماء ويقلب الأنابيب بمعدل بطيء (1 الجولة / ثانية) لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة أنبوب من النمام المغناطيسي مع لوحة التدفئة وترك شظايا العقدة الليمفاوية تسوية. </li><li style=";text-align:right;direction:rtl"> إزالة بعناية طاف المخصب في &quot;خلية غير اللحمية&quot;. ملاحظة: إذا كان تحليل T، B، والخلايا الجذعية وCD45 - gp38 - CD31 - ومن المتوقع، حفظ &quot;الخلايا غير اللحمية&quot; الكسر. </li><li style=";text-align:right;direction:rtl"> غسل الأنسجة المتبقية العقدة الليمفاوية مرة واحدة مع 750 ميكرولتر المتوسطة الأساسي. ملاحظة: هذه الخطوة غير ضرورية إلا إذا كان يعمل مع الفئران محصنة الحكومية المختصة. </li><li style=";text-align:right;direction:rtl"> السماح شظايا العقدة الليمفاوية تسوية. </li><li style=";text-align:right;direction:rtl"> إزالةغير اللحمية جزء العائمة الخلية. </li><li style=";text-align:right;direction:rtl"> أضف إلى شظايا العقدة الليمفاوية 750 ميكرولتر المتوسطة الأساسي تستكمل مع 3.5 ملغ / مل كولاجيناز D و 40 ميكروغرام / مل الدناز I. </li><li style=";text-align:right;direction:rtl"> مكان أنبوب مرة أخرى في الكأس الذي يحتوي على 37 ° C مسخن الماء. </li><li style=";text-align:right;direction:rtl"> هضم الأنسجة الليمفاوية عقدة لمدة 5 دقائق مع التحريك ببطء. </li><li style=";text-align:right;direction:rtl"> تفصيل الأنسجة الليمفاوية شظايا عقدة من قبل pipetting والاختلاط 700 ميكرولتر لمدة 10 دورات في السرعة القصوى باستخدام ماصة الأقنية الآلي. ملاحظة: هذه الأنسجة الليمفاوية يعطل عقدة لتحسين الهضم. </li><li style=";text-align:right;direction:rtl"> وضع أنبوب مرة أخرى في الكأس مع 37 درجة مئوية الماء مسخن. </li><li style=";text-align:right;direction:rtl"> هضم الأنسجة شظايا العقدة الليمفاوية لمدة 10 دقيقة مع التحريك ببطء. </li><li style=";text-align:right;direction:rtl"> تفصيل الأنسجة الليمفاوية شظايا عقدة من قبل pipetting وخلط لمدة 99 دورات في السرعة القصوى باستخدام ماصة الأقنية الآلي. </li><li style=";text-align:right;direction:rtl"> إضافة 7.5 ميكرولتر من 0.5 M EDTA لضمان الحفاظ على تعليق خلية واحدة. </li><li style=";text-align:right;direction:rtl"> تفصيل شظايا الأنسجة العقدة الليمفاوية بواسطة الأنابيبtting وخلط لمدة 99 دورات في السرعة القصوى باستخدام ماصة الأقنية الآلي. </li><li style=";text-align:right;direction:rtl"> إضافة 750 ميكرولتر المتوسطة الأساسي وتمر الخلايا من خلال شبكة من النايلون 70 ميكرون. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي تعليق خلية 5 دقائق عند 1،500 x ج، 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> ويمكن الآن أن تستخدم خلايا انسجة لمزيد من التحليل. </li></ol> 2. تلون الخلايا الليمفاوية عقدة اللحمية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام مزيج من مكافحة CD45، ومكافحة podoplanin (gp38)، والأجسام المضادة لمكافحة CD31 التعرف على الخلايا TRC، بكا، BEC وDN بنجاح. </li><li style=";text-align:right;direction:rtl"> احتضان هضمها العقدة الليمفاوية الأنسجة مع لايف / تعقب الميت، ومكافحة CD45-FITC (1: 200)، ومكافحة gp38-PE (1: 200)، ومكافحة CD31-APC (1: 200) في 100 ميكرولتر HBSS تحتوي على 2 FCS٪ لمدة 20 دقيقة على الأقل، 4 درجات مئوية، في الظلام. </li><li style=";text-align:right;direction:rtl"> غسل الخلايا بإضافة 500 لتر من HBSS تحتوي على 2٪ FCS </li><li style=";text-align:right;direction:rtl"> الطرد المركزي تعليق خلية 3 دقيقة في 1500 x ج، 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> إعادة تعليق في 100 ميكرولتر من HBSS تحتوي على 2٪ FCS. </li><li style=";text-align:right;direction:rtl"> تشغيل الخلايا الملون في تدفق عداد الكريات تجهيز المنطقةإد مع البصريات التالية: مصدر الإثارة مع ما يصل الى ثلاثة أجهزة الليزر: الأزرق (488 نانومتر، وتبريد الهواء، و 20 ميغاواط الحالة الصلبة)، الحمراء (633 نانومتر، 17 ميغاواط الحنة)، والبنفسجي (405 نانومتر، 30 ميغاواط الحالة الصلبة) . </li><li style=";text-align:right;direction:rtl"> بوابة CD45 - خلايا لاستبعاد الخلايا المكونة للدم. </li><li style=";text-align:right;direction:rtl"> singlets بوابة (FSC-W) والخلايا الحية (لايف / تعقب الميت) لاستبعاد الحلل والخلايا الميتة. </li><li style=";text-align:right;direction:rtl"> مؤامرة gp38 مقابل CD31 لتصور TRC (gp38 + CD31 -)، كرا (gp38 + CD31 +)، BEC (gp38 - CD31 +) وDN (gp38 - CD31 -) الخلايا (انظر الشكل 1). </li></ol>

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The authors declare they have no competing financial interests.

دراسة خلايا انسجة العقدة الليمفاوية أصبحت في الآونة الأخيرة التركيز على البحوث نظرا لتطوير بروتوكولين الهضم نشرت 6،13. كلا البروتوكولين كافية للحصول على الخلايا الليمفاوية واحد العقدة انسجة ولكنها تختلف في استخدام إنزيمات الهضم ووقت الهضم. منذ خلايا انسجة وعل...

الغدد الليمفاوية هي مقصورات المتخصصة حيث بدأت الاستجابات المناعية التكيفية ضد المستضدات الذاتية والخارجية ومنسقة. الإجراء المقدمة هنا يصف الهضم الأنزيمي قصيرة جنبا إلى جنب مع pipetting لالميكانيكي الآلي للحصول على العقدة اللمفاوية وحيدة الخلية تعليق والوصول إلى خلايا انسجة العقدة الليمفاوية قابلة للحياة التي تحافظ على التعبير السطح العديد من الجزيئات. الخلايا اللحمية العقدة الليمفاوية تشكل سقالة من العقدة الليمفاوية والوفاء ثلاث وظائف رئيسية: أولا أنها تصفية سوائل الجسم لأخذ عينات المستضدات، ومسببات الأمراض والعوامل المسببة للأمراض المرتبطة نمط الجزيئية الخاصة بهم (PAMPs)، وكذلك السيتوكينات والخطر المرتبطة نمط الجزيئية (DAMPs) الحاضر في الجسم. ثانيا، جذب وإرشاد مستضد تقديم الخلايا (APC) والخلايا الليمفاوية للتفاعل والشروع في الاستجابات المناعية التكيفية. وثالثا، لأنها توفر بيئة الهيكلية للتوازن وتمايز الخلايا الليمفاوية 1-3. أثناء التهاب خلايا العقدة الليمفاوية انسجة تنتج عوامل النمو، السيتوكينات وكيموكينات، والتكيف مع تورم وبالتالي تنظيم التفاعل بين الخلايا الجذعية (DCS)، تي وخلايا ب. تزامن الاستجابات المناعية ممكن فقط بسبب بنية الهيكلية المعقدة التي شكلتها مختلف السكان الخلية اللحمية. خلايا انسجة العقدة الليمفاوية هي خلايا CD45 السلبية ويمكن تمييزها عن طريق التعبير عن CD31 أو gp38 في خلايا ليفية والبطانية 1 -6. Gp38 + CD31 - يحدد خلايا T منطقة شبكي (TRC، والتي تعرف أيضا باسم FRC: خلايا الشبكية ليفية)، gp38 + CD31 + الخلايا الليمفاوية يعرف البطانية (كرا)، gp38 - CD31 + يحدد الخلايا البطانية في الدم (BEC). علاوة على ذلك، توصيف المجموعات السكانية الفرعية وكشف وجود خلايا انسجة العقدة الليمفاوية الأخرى. في الواقع، واتسم عدد صغير من السكان خلية تشبه خلية حوطية داخلgp38 - CD31 - السكان 7. لذا، والتكيف من إجراء العزل هو مفيد لتحديد وتوصيف خصائص وظيفية مختلفة من خلايا انسجة العقدة الليمفاوية. قبل وضع العقدة الليمفاوية خلايا انسجة الهضم البروتوكولات دراسة خلايا انسجة العقدة الليمفاوية اقتصر على الرصدات في الموقع باستخدام قسم الأنسجة والفحص المجهري. ومع ذلك، أظهرت الدراسات البنيوية والوظيفية الخصائص المهمة للخلايا انسجة العقدة الليمفاوية. وترتبط خلايا انسجة العقدة الليمفاوية مع podoplanin، والكولاجين والمصفوفة خارج الخلية (ECM) البروتينات لتشكيل بنية معقدة 3 الأبعاد يسمى نظام القناة الذي ينقل الليمفاوية وما يرتبط بها منخفضة الكتلة الجزيئية البروتينات من الجيوب تحت المحفظة من العقدة الليمفاوية إلى البطانية عالية الأوردة في منطقة خلايا T 8. البلدان النامية على اتصال وثيق مع خلايا سدى ويمكن ملاحظة جاحظ في يخدع أنبوبيهيكل duit لأخذ عينات السوائل وكشف المستضدات 8. وبوساطة تفاعل خلايا انسجة العقدة الليمفاوية (TRCS وموظفين المدنيين المحليين) مع البلدان النامية من خلال الإفراج وعرض كيموكينات CCL21 و CCL19 9،10. يتم الاعتراف CCL19 وCCL21 بواسطة مستقبلات CCR7 تسهيل البلدان النامية والخلايا T للهجرة إلى منطقة العقدة الليمفاوية الخلية T 4،11. على الرغم من استخدام كيموكينات مماثلة، وخلايا T البلدان النامية ولها طرق الهجرة المختلفة في الغدد الليمفاوية 12. في وقت لاحق، وذلك باستخدام الهضم الأنزيمي من العقدة الليمفاوية وعزل الخلايا اللحمية العقدة الليمفاوية نقية، وأجريت دراسات وظيفية على دور مختلف خلايا العقدة الليمفاوية اللحمية وقدرتها على التفاعل مع البلدان النامية وخلايا T / B 6،13. أولا، الحديث المتبادل بين IFN-γ إنتاج خلايا T المستجيب والخلايا اللحمية العقدة الليمفاوية الحث على إنتاج أكسيد النيتريك المستقلب تبين أن يثبط استجابات الخلايا T والانتشار في أجهزة اللمفاوية الثانوية 14-16. ثانيا، الليمفاوية نوقد تم الإبلاغ عن خلايا انسجة صدى لدعم التفريق بين مجموعات فرعية DC التنظيمية عبر إنتاج IL-10 17، والسذاجة لتعديل توازن الخلايا التائية عبر إنتاج IL-7 6،18. ثالثا، TLR التعبير في خلايا انسجة العقدة الليمفاوية تشير إلى أن خلايا انسجة عرضة للإشارة مشتقة من عدوى أو الجزيئات الذاتية صدر خلال إصابة الأنسجة. والواقع أن علاج الخلايا اللحمية العقدة الليمفاوية مع يجند من TLR3 بولي (I: C) يؤدي الى upregulation متواضع من التوافق النسيجي الرئيسي التعبير الدرجة الأولى ومعقدة upregulation المشترك المثبطة جزيء PD-L1، ولكن ليس من الجزيئات costimulatory، مما أدى إلى تغييرات جذرية في الأنسجة الطرفية مستضدات التعبير 19. وقد أظهرت عدة مجموعات خلايا انسجة العقدة الليمفاوية التعبير عن مستضدات الأنسجة الطرفية وتحفز التسامح من ذاتية التفاعل خلايا T 19،21-27. لذا، فهم التفاعلات بين الخلايا اللحمية العقدة الليمفاوية وغيرها من إعادة المهاجرة وسوف المنشقة خلايا العقدة اللمفاوية يساعد على إيجاد الجزيئات المستهدفة الجديدة للسماح تفعيل أو قمع الاستجابات المناعية أثناء الالتهاب. ولذلك، هناك حاجة إلى تنفيذ الفصل الأنزيمية نشرت من العقدة الليمفاوية. البروتوكولات نشرت سابقا استخدام تركيبات مختلفة من القائم كولاجيناز الأنزيمية الهضم مع انخفاض الضغط الميكانيكي 6،19،20. ومع ذلك، حضانات طويلة مع إنزيمات الهضم أو مزيج مختلف من انزيم الهضم قد تتحلل مختلف الجزيئات السطحية اللازمة لتحليل حالة التنشيط وتحديد خلايا انسجة العقدة الليمفاوية جديدة. اعتمادا على نوع من التحليل الخلايا اللحمية، وبروتوكول وصلة أو بروتوكول فليتشر قد يكون أكثر ملاءمة. في الإجراء وصفها، يتم الجمع بين الهضم الأنزيمي أقصر قليلا مع تصنيف الميكانيكي الآلي للحد من تدهور سطح علامة قابلة للحياة الخلايا الليمفاوية من عقدة اللحمية. يتيح هذا الإجراء العزلة استنساخه للغاية وتمييز من العقدة الليمفاوية السكان الخلية اللحمية مع تقلب منخفض وأكثر من 95٪ جدوى. الخلايا اللحمية العقدة الليمفاوية المعزولة حديثا يمكن استخدامها مباشرة للتعبير عن علامة السطح، وتحليل البروتين، والدراسات النسخي، وكذلك إنشاء خطوط خلايا انسجة لإجراء المقايسات الفنية في المختبر.

<table border="0" cellpadding="0" cellspacing="0" width="959">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:231px;">LifeTechnologies-Gibco</td>
			<td style="width:119px;">41965-039</td>
			<td style="width:395px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FCS</td>
			<td style="width:231px;"></td>
			<td style="width:119px;"></td>
			<td>Final concentration 2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">499609</td>
			<td>Final concentration 1.2 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Collagenase IV</td>
			<td style="width:231px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;"></td>
			<td>&#62;160 units per mg dry weight, use at final concentration of 1 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:231px;">Roche</td>
			<td style="width:119px;">11088882001</td>
			<td>use at 3.5 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNAse I</td>
			<td style="width:231px;">Roche&#160;</td>
			<td style="width:119px;">11284932001</td>
			<td>use at 40 &#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stiring magnets</td>
			<td style="width:231px;">FAUST</td>
			<td style="width:119px;"></td>
			<td>5 mm long-2 mm &#248;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polystyren Round-Bottom Tubes 5ml</td>
			<td style="width:231px;">Falcon-BD Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnetic stirrer with heating funktion</td>
			<td style="width:231px;">IKA-RCT-standard</td>
			<td style="width:119px;">9720250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petridishes 100 mm, sterile</td>
			<td style="width:231px;">TPP</td>
			<td style="width:119px;">6223201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needles</td>
			<td style="width:231px;">Terumo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD45 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 30-F11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD11c Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone N418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse Podoplanin Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 8.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD31 Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone MEC13.3</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;"> 
			Eppendorf Xplorer plus, Multichannel</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:119px;">4861 000.821/830</td>
			<td>1.250 &#181;l max. volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD140a</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone APA5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD80</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 16-10A1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD40</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 1C10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse I-Ab</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone AF6-120.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti mouse CD274 (PD-L1)</td>
			<td style="width:231px;">Biolegend</td>
			<td style="width:119px;"></td>
			<td>Clone 10F.9G2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Near-IR Dead Cell stain kit</td>
			<td style="width:231px;">Invitrogen</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

هذا البروتوكول هو بروتوكول الهضم تعديل نشرتها رابط وآخرون، 2007 6 مع أقصر وقت الهضم (45 دقيقة كحد أقصى) بسبب التقسيم الميكانيكية مع ماصة الأقنية الآلي. بالإضافة إلى ذلك، هذا الإجراء هو أكثر توحيدا، ويقلل من تدهور علامات على سطح خلايا انسجة مختلفة العقدة الليم?...

Watch this Scientific Journal Video about Isolation of Murine Lymph Node Stromal Cells at JoVE.com

Isolation of Murine Lymph Node Stromal Cells

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 ...

isolation-of-murine-lymph-node-stromal-cells

Research

JoVE Journal

Immunology and Infection

30.9K Views.  University of Basel and University Hospital Basel. 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. 

Video: Isolation of Murine Lymph Node Stromal Cells

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 ...

Murine Superficial Lymph Node Surgery

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 followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

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 ...

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 ...

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 ...

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, ...

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

عزل مجموعات الضامة والخلايا اللحمية من الإنسان والماوس عينات عضله القلب

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.