نشكر الدكاترة. اليكس Gelbard ويليم Overwijk لتقديم المشورة بشأن نقل الجينات الهيدروديناميكية. وأيد هذا العمل من المنح المقدمة من المعاهد الوطنية للصحة (AI098099، الرياح)، ومركز إم دي أندرسون للسرطان علم التخلق، ومركز أندرسون MD للالتهاب والسرطان (SSW)، وصندوق RE بوب سميث التعليم (الأعرج).

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الكتاب تعلن أي المصالح المالية المتنافسة.

تقنية نقل بالتبني وصفها المقررة في هذه الوثيقة مساهمة CDPs للسكان PP PDC في الفئران المتلقية التي تعاني من نقص في PP pDCs (على سبيل المثال Ifnar - / - الفئران). في التجارب في المستقبل، سيكون من المهم لتقييم إمكانات مجموعات فرعية السلف الأخرى في توليد pDCs PP، وبخاصة ما إذا ...

هنا، علينا أن نظهر بروتوكول لتقييم ما إذا كانت الخلايا الجذعية الأسلاف المشتركة (CDPs) قادرة على التسبب في الخلية الجذعية بلازماوية (PDC) السكان في التصحيح باير (PP). وكان الهدف العام من استخدام هذا الأسلوب لتقييم التنظيم التنموية للpDCs في التصحيح باير (PP pDCs). وسبب هذا هو المهم هو أن pDCs PP تختلف عن pDCs وجدت في الأنسجة الأخرى، بما في ذلك نخاع العظام والدم والطحال، وبالتالي فإنه من غير الواضح ما إذا كان pDCs PP والسكان PDC الأخرى تنمويا و / أو ذات الصلة وظيفيا. على وجه التحديد، ومن المعروف على نطاق واسع pDCs لكونها نوع الرئيسي أنا إنترفيرون (الإنترفيرون) المنتجين داخل منظومة المكونة للدم، والاستجابة لتول مثل مستقبلات 7 و 9 (TLR7 / 9) عن طريق تحفيز إفراز الإنترفيرون السريع 1-3. ومع ذلك، pDCs PP تعاني من نقص في إنتاج النوع الأول الإنترفيرون ردا على TLR التحفيز ناهض 4،5. علاوة على ذلك، PP pDCs أيضا تختلف عن pDCs وجدت في نخاع العظام والطحال فيتتطلب إشارات من نوع I إنترفيرون (الإنترفيرون) مستقبلات (IFNAR1) أو الإنترفيرون يشير STAT1 جزيء لتنميتها و / أو تراكم 5. وقد اقترح هذه البيانات إمكانية أن آليات تنظيمية واضحة تحكم pDCs PP مقابل pDCs في الأجهزة الأخرى (مثل نخاع العظم والطحال) 5. واستند الأساس المنطقي الذي أدى إلى تطوير هذا الأسلوب على التطورات الحديثة في فهم الخلايا الجذعية (DC) علم الأحياء. معظم، إن لم يكن كلها، DC مجموعات فرعية مستمدة من الأسلاف المكونة للدم التي تعبر عن FMS مثل التيروزين كيناز 3 مستقبلات (FLT3) 6-10، ولكن لا يقتصر DC التنمية إلى النخاعي الكلاسيكية والممرات اللمفاوية. على سبيل المثال، FLT3 + الأسلاف النخاعي المشتركة (CMPS، لين - IL-7R - هيئة السلع التموينية-1 - ج + CD34 + عدة FcγR الصغرى / -) تؤدي إلى CDPs (لين - ج طقم لو CD115 + + FLT3)، whicح تمييز الى مزيد من pDCs والبلدان النامية التقليدية (CDCS) 9،10. على النقيض من ذلك، FLT3 + الأسلاف اللمفاوية المشتركة (CLPS، لين - IL-7R + هيئة السلع التموينية-1 لو ج طقم الصغرى) في المقام الأول إلى تطوير pDCs 11. لذلك، تشير دراسات سابقة تنشأ من pDCs لا يقل عن 2 متميزة السكان الاصلية المكونة للدم تحت تنظيم Flt3L، على الرغم من أن تحليل نموذجي وقد يقتصر على نخاع العظم والطحال و / أو مجموعات فرعية PDC الدم. وبالتالي، فإن عدد سكان السلف (ق) التي يولد PP pDCs المطلوب التحقيق. وفهم أصول PP pDCs تسليط الضوء على ما إذا كانت مشاركة مسارات تنموية مشتركة مع السكان PDC أخرى، أو استخدام آليات متميزة خلال جيلهم في مؤتمر المندوبين المفوضين. وهناك ميزة فريدة من النهج الموصوفة هنا هو استخدام الفئران التي تظهر نقص حاد في PP pDCs كمتلقين لنقل بالتبني من الأسلاف المكونة للدم. الفئران مع الجيناتحذف التشنج في ترميز الجين IFNAR1 (Ifnar - / - الفئران) أو STAT1 (STAT1 - / -) كشفت عن وجود استنزاف لافتة في PP pDCs 5. لذلك، توفر هذه السلالات بيئة التي يتم فيها تخفيض pDCs PP، مما يتيح نقل بالتبني الدراسات التي يتعين القيام بها في غياب الأنظمة الاجتثاث خلية قوية مثل أشعة قاتلة. وقوة إضافية من الطريقة المعروضة هنا هو استخدام نقل الجينات الهيدروديناميكية (HGT) لتحفيز كميات مرتفعة من تعميم Flt3L. هذا يوفر نهجا فعالة من حيث التكلفة للحث على Flt3L في الجسم الحي، مقابل حقن البروتين المؤتلف. وقد استخدمت العديد من الدراسات، بما في ذلك تلك الموجودة في مختبرنا، HGT للحث على كميات خلوى في مجموعة متنوعة من الظروف التجريبية 5،12،13. تقسيم العمل وظائف المناعة دقيقة بالنسبة للبلدان النامية هو من مصلحة كبرى في علم المناعة. على وجه الخصوص، pDCs وسطاء المهم التسامح عن طريق الفم والنظامية المضادة للفيروساتالاستجابات، ولكنها تظهر أيضا على المساهمة في التنمية واستمرار المناعة الذاتية والسرطان 14-17. سوف بروتوكول الموصوفة هنا تسمح الآليات التنموية التي تنظم PP pDCs أن يكون استكشاف أكثر اكتمالا. بالإضافة إلى ذلك، قد يسمح هذا النهج من الدراسات لتقييم وظيفة PP PDC، ويجوز تمديدها لفهم التنظيم وظيفة من السكان المناعية الأخرى داخل ذكر المكتب الصحفى.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">C57BL/6J</td>
			<td style="width:201px;">JAX</td>
			<td style="width:177px;">664</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">B6.SJL</td>
			<td style="width:201px;">JAX</td>
			<td style="width:177px;">2014</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RPMI</td>
			<td style="width:201px;">Invitrogen</td>
			<td style="width:177px;">11875-093</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">15 ml conical tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352095</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">50 ml conical tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352070</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile surgical tweezers</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile small pair scissors</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Sterile large pair scissors</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">40 &#956;m cell strainer</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352340</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">35 &#956;m cell strainer cap tubes</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">352235</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">RBC lysing buffer</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:177px;">R7757</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:260px;">FACS buffer</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;">PBS, 2 mM EDTA, 1% FCS, filter sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Percoll</td>
			<td style="width:201px;">GE Healthcare</td>
			<td style="width:177px;">17089102</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">10XHBSS</td>
			<td style="width:201px;">Sigma</td>
			<td style="width:177px;">H4641</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Collagenase IV</td>
			<td style="width:201px;">Worthington</td>
			<td style="width:177px;">LS004188</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Goat ant-rat IgG microbeads</td>
			<td style="width:201px;">Milteyni Biotec</td>
			<td style="width:177px;">130-048-501</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">LD column</td>
			<td style="width:201px;">Milteyni Biotec</td>
			<td style="width:177px;">130-042-901</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-D3</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">555273</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD19</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553783</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD11b</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553308</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-CD11c</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553799</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Rat anti-Ter119</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">553671</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD3 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0031</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD19 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0193</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11b (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0112</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11c (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-0114</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-F4/80 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-4801</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Ter119 (PerCP)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">45-5921</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Sca-1 (PE.Cy7)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">25-5981</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD115 (APC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">17-1152</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-c-kit (APC.Cy7)&#160;</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">47-1171</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-IL-7R (Pacific Blue)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">48-1271</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Flt3 (PE)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">12-1351</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD45.1 (APC.Cy7)</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;">560579</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD45.2 (PE.Cy7)</td>
			<td style="width:201px;">Biolegend</td>
			<td style="width:177px;">109830</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-CD11c (Pacific Blue)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">48-0114</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-B220 (APC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">25-0452</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-Siglec-H (PE)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">12-0333</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Anti-PDCA-1 (FITC)</td>
			<td style="width:201px;">eBiosciences</td>
			<td style="width:177px;">Nov-72</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Cell sorter</td>
			<td style="width:201px;">BD Biosciences</td>
			<td style="width:177px;"></td>
			<td style="width:229px;">e.g. BD Fortessa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Heat lamp</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">Mouse restrainer</td>
			<td style="width:201px;"></td>
			<td style="width:177px;"></td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">1 ml syringes</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td style="width:177px;">309602</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:260px;">27&#189; G needles (sterile)</td>
			<td style="width:201px;">Becton Dickinson</td>
			<td style="width:177px;">305109</td>
			<td style="width:229px;"></td>
		</tr>
	</tbody>
</table>

assessing the development of murine plasmacytoid dendritic cells in peyer's patches using adoptive transfer of hematopoietic progenitors

هذا البروتوكول تفاصيل طريقة لتحليل قدرة الأسلاف المكونة للدم النقي لتوليد الخلايا الجذعية بلازماوية (PDC) في الأمعاء التصحيح باير (PP). وتنقيته من نخاع العظام من الفئران C57BL6 بواسطة نظام مراقبة الأصول الميدانية ونقلها إلى المتلقي الفئران التي تفتقر إلى السكان PDC كبيرة في PP - الأسلاف المشتركة الخلايا الجذعية (ج طقم لو CD115 + + FLT3 CDPs، لين)، وفي هذه الحالة، Ifnar - / - استخدمت الفئران كما المتلقين نقل. في بعض الفئران، وقد تم فرض overexpression من وعامل نمو الخلايا الجذعية FLT3 يجند (Flt3L) قبل بالتبني نقل CDPs، وذلك باستخدام نقل الجينات الهيدروديناميكية (HGT) من Flt3L ترميز البلازميد. Flt3L من overexpression يوسع سكان العاصمة مصدرها نقل (أو الذاتية) الأسلاف المكونة للدم. في 7-10 أيام بعد نقل السلف، تميزوا pDCs التي تنشأ عن الأسلاف نقل adoptively من خلايا المتلقي علىأساس CD45 علامة التعبير، مع pDCs من CDPs نقل يجري CD45.1 + والمتلقين يجري CD45.2 +. تم تقييم قدرة CDPs نقلها إلى المساهمة في السكان PDC في PP والرد على Flt3L بواسطة التدفق الخلوي من PP تعليق خلية واحدة من الفئران المتلقية. هذه الطريقة يمكن استخدامها لاختبار ما إذا كان السكان السلف الأخرى قادرة على توليد PP pDCs. بالإضافة إلى ذلك، يمكن استخدام هذا النهج لدراسة دور العوامل التي من المتوقع أن تؤثر على التنمية PDC في PP، عن طريق نقل مجموعات فرعية السلف مع ضربة قاضية، أو خروج المغلوب من overexpression المناسبة للعامل التنموية المفترضة و / أو عن طريق التلاعب السيتوكينات المتداولة عبر HGT . هذه الطريقة قد تسمح أيضا تحليل الكيفية التي تؤثر pDCs PP تردد أو وظيفة فرعية المناعية الأخرى في ذكر المكتب الصحفى. ومن المزايا الفريدة لهذا الأسلوب هو استخدام Ifnar - / - الفئران، والتي تظهر استنزاف شديد PP pDCs نسبة إلى نوع الحيوانات البرية، وبالتالي allowiنانوغرام إعادة تشكيل PP pDCs في غياب آثار الخلط من أشعة قاتلة.

نتائجنا تظهر استراتيجية النابضة لعزل CDPs من الماوس خلايا نخاع العظم (الشكل 1)، على النحو المفصل في البروتوكولات 2 و 3. تشمل CDPs حوالي 0.1٪ من مجموع خلايا نخاع العظام، وحوالي 4-6 × 10 4 CDPs يمكن عزلها عن الماوس واحد. عند نقل بالتبني، CDPs تفرق في pDCs وCDCS 10. <p clas...

Watch this Scientific Journal Video about Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors at JoVE.com

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

يصف هذا البروتوكول إجراءات تجريبية لتقييم تمايز الخلايا الجذعية بلازماوية في التصحيح باير الأسلاف من الخلايا الجذعية المشتركة، وذلك باستخدام التقنيات التي تنطوي على نظام مراقبة الأصول الميدانية بوساطة العزلة الخلية، ونقل الجينات الهيدروديناميكية، وتحليل تدفق مجموعات فرعية المناعة في التصحيح باير.

تقييم تطوير خلايا الفئران الجذعية بلازماوية في بقع باير طريق نقل بالتبني من أسلاف المكونة للدم

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

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Research

JoVE Journal

Immunology and Infection

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

11.1K Views.  The University of Texas MD Anderson Cancer Center.  يصف هذا البروتوكول إجراءات تجريبية لتقييم تمايز الخلايا الجذعية بلازماوية في التصحيح باير الأسلاف من الخلايا الجذعية المشتركة، وذلك باستخدام التقنيات التي تنطوي على نظام مراقبة الأصول الميدانية بوساطة العزلة الخلية، ونقل الجينات الهيد...

Video: Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

الإنبثاث التجريبية وCTL المناعي نقل بالتبني النموذجي ماوس

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

عزل والخلايا اللمفاوية المناعية والخلايا الجذعية من الفئران باير لالرقع

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

المعجل نوع 1 الحث السكري في الفئران عن طريق نقل بالتبني من CD4 + T خلايا سكري

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

تقييم الفطرية الاستشعار من HIV-1 CD4 المصابة<sup&gt; +</sup&gt; T الخلايا عن طريق الخلايا الجذعية بلازماوية الشكل باستخدام<em&gt; خارج الحي</em&gt; المشارك ثقافة النظام.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

تحريض الفئران المعوية التهاب عن طريق نقل بالتبني من المستجيب CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; عالية</sup&gt; خلايا T في الفئران العوز المناعي

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

في المختبر جيل من الخلايا الجذعية من الفئران بلازماوية الشكل المشتركة اللمفاوية الأسلاف باستخدام نظام تغذية AC-6

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

مضان تنشيط الفرز الخليوي لتنقية الخلايا الجذعية بلازماوية الشكل من الفأر نخاع العظم

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

توليد عدد كبير من فروعه النقوي وسلائف الخلايا الجذعية من نخاع العظام مورين باستخدام خلية رواية فرز استراتيجية

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

التنمية والتحقق من جزيء واحد أولتراسينسيتيفي الصفيف الرقمي المرتبط بالانزيم مقايسة الممتز للبشرية انترفيرون-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

نقل الخلايا المناعية السامة للخلايا في نماذج مورين لتكثيفها عديدة الأشكال جليوبلاستوما أورثوتوبيك الإنسان التبني المناظير باستخدام الأشعة

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...