This work was supported by EU FP7-ITN Marie-Curie Network program RADDEL. MB and KAJ would like to acknowledge Joe Varguese and Rui Serra Maia for their help and suggestions along the optimization of the Kupffer cell isolation.

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+cells+from+livers+of+carcinogen-treated+rats+by+free-flow+electrophoresis.">Purification of cells from livers of carcinogen-treated rats by free-flow electrophoresis.</a> Cancer Res. 43 (9), 4176-4179 (1983).</li><li>Firme, C. P., Bandaru, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+issues+in+the+application+of+carbon+nanotubes+to+biological+systems.">Toxicity issues in the application of carbon nanotubes to biological systems.</a> Nanomedicine. 6 (2), 245-256 (2010).</li><li>Ali-Boucetta, H., Al-Jamal, K. 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The authors declare that they have no competing financial interests.

الخطوات التالية هي حاسمة لتحقيق عائدات عالية وقابلية عالية للخلايا كوبفر. وينبغي أن تستخدم ظروف معقمة للحد من مخاطر التلوث الجرثومي والفطري. يتعين على جميع الأدوات اللازمة لتعقيمها قبل استخدامها. الكواشف يجب الطازجة قبل تنفيذ إجراء العزل. <p class="jove_content" style=";text-align:r...

ويهدف مجال البحث nanotoxicology لوصف التأثير البيولوجي للجزيئات النانوية. لا تزال الدراسات السمية بناء على التحقيقات المجراة الطرق الأكثر دقة. ومع ذلك، فإن استخدامها محدود بسبب التكلفة والعمل والوقت متطلباتهم 1. كما تم استخدام البدائل، فحوصات في المختبر بسبب بساطتها وإمكانية تطوير الإنتاجية العالية في منصات اختبار المختبر 2 - حتى تمديد عدد من الشروط التي تم اختبارها. يتم تنفيذ معظم الدراسات nanotoxicological استخدام في فحوصات المختبر مع خطوط الخلايا خلد. ومع ذلك، هناك مخاوف بشأن استقراء هذه النتائج التجريبية لفي الجسم الحي الآثار السمية 3. في الواقع، يمكن أن خصائص خطوط الخلايا خلد تكون مختلفة كثيرا عن الأنسجة كانوا المتأتي من مثل التحول الجيني 4، وتدهور الخصائص المورفولوجية الرئيسية5، وفقدان قطبية الخلوية 6 والتعديلات الفنية مثل تنظيم وسطاء التهابات 7. خلايا كوبفر هي السكان بلعم الأكثر وفرة في الجسم، وهي على اتصال مباشر مع الدم عن طريق المبطنة لجدار الجيوب الكبد. كجزء من الجهاز الشبكي البطاني (RES)، وهذه البلاعم هي المسؤولة عن القبض على تعميم النانوية، وبالتالي، تشكل نموذجا مناسبا للغاية لدراسة سمية جسيمات متناهية الصغر. في الجسم الحي 8 وفي المختبر وقد نشرت دراسات 9 من الاستجابات الالتهابية المرتبطة مع خلايا كوبفر تتعرض لالنانوية في مكان آخر. كما شاركت خلايا كوبفر في التسبب في أمراض الكبد مثل أمراض الكبد الكحولية 10، تليف الكبد أو التهاب الكبد الفيروسي 11 12. وأفيد أن خلايا كوبفر معزولة توفر البصيرة مفيدة لوصف الآليات الخلوية الجردolved في اضطراب الكبد 13،14. وقد تم الإبلاغ عن عدة طرق لعزل وتنقية خلايا كوبفر. عزل الخلايا يمكن أن تنجم عن ميكانيكي أو الأنزيمية التفكك 15. يظهر الهضم كولاجيناز ميزة الحفاظ على سلامة الوظيفية للخلايا كوبفر، في حين يؤدي إلى ارتفاع كوبفر الخلايا العائد 16. مناهج تجريبية عديدة، متفاوتة في التعقيد والتكاليف، وقد استخدمت لفصل خلايا كوبفر من غيرهم من السكان خلايا الكبد. على سبيل المثال، لا يمكن أن يتحقق كوبفر نقاء الخلية immunoaffinity 17، وتدفق الكهربائي 18، التصاق الانتقائي 16 أو من خلال تقنيات الطرد المركزي 16، والذي حدد الخلايا وفقا لحجمها وكثافة. مزيج من هذه الطرق يمكن اختيار لزيادة نقاء من السكان 16. لا يوجد إجماع حول الأسلوب الأمثل لعزل الخلايا كوبفر، لأنها تعتمد في الغالب على تطبيق والمعدات المتاحةر. ومع ذلك، في حالة اختبار السمية جسيمات متناهية الصغر، وبساطة وإنتاجية عالية من التقنية المرتبطة الحفاظ الوظيفي للخلايا كوبفر يبدو أن الأكثر ملاءمة لهذا التطبيق. ويستند كوبفر طريقة عزل الخلايا ذكرت هنا على طريقة التروية 2-خطوة تليها تنقية على التدرج الكثافة. تم تعديل طريقة من البروتوكول الأصلي الذي وضعته Smedsrød وآخرون 16 تصميم لعزل خلايا كبد الفئران. ذكرت معظم الدراسات وصفت عزل خلايا كوبفر من كبد الفئران. هنا، نحن تصف طريقة لعزل خلايا كوبفر من كبد الفأر، في ارتفاع العائد والنقاء. استخدام الفئران يقلل من تكلفة التجربة ويسمح بتجهيز عدة كبد للحصول على كميات كبيرة من خلايا كوبفر في اختبارات السمية جسيمات متناهية الصغر. في بروتوكول التالية، وحضنت خلايا كوبفر مع أنابيب الكربون النانوية functionalized (و </م&gt; -CNTs). الخصائص الفيزيائية والكيميائية الفريدة من الأنابيب النانوية الكربونية - أي طول ارتفاع نسبة القطر ومساحة كبيرة، جعلت الأنابيب النانوية الكربونية المرشحين للاهتمام وهي الحاملة لأغراض العلاج والتشخيص. ومع ذلك، فقد أثيرت مخاوف بشأن سمية تشارك المركز الوطني 19، وتطوير جديدة في اختبارات المختبر يهدف إلى زيادة فهم تأثير بيولوجي CNT. ويرتبط سمية في خلية كوبفر مع عدم وجود السلامة الهيكلية للغشاء الخلية. يتم قياس ذلك من خلال فقدان إنزيم LDH حشوية من الخلية إلى طاف. مبدأ هذه الطريقة، لذلك، هو إزالة أي LDH صدر وقياس ما تبقى في خلايا 20. ويتم ذلك في الأفضلية لقياس LDH صدر في طاف لأن وجود الأنابيب النانوية الكربونية في طاف يتداخل مع مقايسة 21. نقترح استخدام هذا فعال metho كوبفر العزلة خلية بسيطة والتكلفةد لعزل عدد كبير من خلايا كوبفر وظيفية. وهذا يسمح للفحص السمية من مجموعة من الجسيمات النانوية، في نموذج بلعم الأولية ذات الصلة.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td dir="LTR">Euthatal (pentobarbital sodium)</td>
			<td dir="LTR">Merial</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
		</tr>
		<tr>
			<td dir="LTR">CD1 mice</td>
			<td dir="LTR">Charles River&#160;&#160;</td>
			<td dir="LTR">-</td>
			<td dir="LTR">Mouse weight should vary from 35 to 45 g. We advise the use of male CD1 mice as their weight increase rapidly (e.g. a male CD1 mouse of 7 weeks old exceeds 35 g in weight). It is also advised to contact the animal supplier in advance to arrange for the delivery of older animals. Alternatively animals can be in-house to reach the desired weight.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">HBSS (Ca2+ and Mg2+ free, with bicarbonate)&#160;&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">14175-053</td>
			<td dir="LTR">HBSS must be&#160; Ca2+ free.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Ethylene Glycol Tetraacetic Acid (EGTA) tetrasodium salt&#160;</td>
			<td dir="LTR">Sigma-Aldrich&#160;&#160;</td>
			<td dir="LTR">E8145&#160;&#160;</td>
			<td dir="LTR">EDTA can also be used but EGTA has the advantage of chelating Ca2+ selectively.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">HEPES (1M)&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">15630-056&#160;</td>
			<td dir="LTR">100 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Collagenase type IV&#160;</td>
			<td dir="LTR">Worthington&#160;</td>
			<td dir="LTR">CSL-4&#160;</td>
			<td dir="LTR">It is advised to test different batches of collagenase or at least mention to the supplier the product has to be suitable for liver cell isolation&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">&#160;Low glucose DMEM&#160;</td>
			<td dir="LTR">Sigma-Aldrich&#160;&#160;</td>
			<td dir="LTR">D5523&#160;</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">RPMI (with sodium pyruvate and Glutamax&#174;)&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">12633-012&#160;</td>
			<td dir="LTR">500 ml</td>
		</tr>
		<tr>
			<td dir="LTR">Penicillin/Steptomycin&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">15140-122</td>
			<td dir="LTR">100 ml</td>
		</tr>
		<tr>
			<td dir="LTR">Fetal Bovine Serum&#160;</td>
			<td dir="LTR">First-Link&#160;&#160;</td>
			<td dir="LTR">60-00-850&#160;</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Trypan blue solution&#160;</td>
			<td dir="LTR">Sigma-Aldrich&#160;</td>
			<td dir="LTR">T8154&#160;</td>
			<td dir="LTR">100 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Coated silica particle solution (Percoll&#174;)</td>
			<td dir="LTR">GE Healtcare&#160;&#160;</td>
			<td dir="LTR">17-0891-02&#160;</td>
			<td dir="LTR">Percoll&#174; is very stable and can be kept for several years&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">1x Phosphate Buffered Saline&#160;&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">10010-023</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">10x Phosphate Buffered Saline&#160;&#160;</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">70011-036</td>
			<td dir="LTR">500 ml</td>
		</tr>
		<tr>
			<td dir="LTR">Dimethyl sulfoxide&#160;</td>
			<td dir="LTR">Fisher&#160;</td>
			<td dir="LTR">D/4120/PB08&#160;</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">LDH kit (CytoTox 96&#174;)&#160;</td>
			<td dir="LTR">Promega&#160;</td>
			<td dir="LTR">G1781&#160;</td>
			<td dir="LTR">Keep protected from light&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">DMEM phenol red free</td>
			<td dir="LTR">Life Technologies&#160;&#160;</td>
			<td dir="LTR">31053-028&#160;</td>
			<td dir="LTR">500 ml&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">F4/80 antibody (Alexa 488)</td>
			<td dir="LTR">AbD Serotec</td>
			<td dir="LTR">MCA497A488</td>
			<td dir="LTR">Do not dilute, used neat for flow cytometry</td>
		</tr>
		<tr>
			<td dir="LTR">Fluorescent beads</td>
			<td dir="LTR">Sigma</td>
			<td dir="LTR">L2778</td>
			<td dir="LTR">Latex beads, amine-modified polystyrene, fluorescent red. 1 ml</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name of the Material</td>
			<td>Company&#160;</td>
			<td>Catalog number&#160;</td>
			<td>Comments/Description&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Butterfly blood collection set (23G/305mm long tubing)&#160;&#160;</td>
			<td dir="LTR">BD&#160;</td>
			<td dir="LTR">367288</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Syringe Filters (0.22 &#956;m Blue Rim)&#160;</td>
			<td dir="LTR">Minisart&#160;</td>
			<td dir="LTR">16534-K&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Centrifuge Tubes (50 ml Blue Cap)&#160;</td>
			<td dir="LTR">BD Biosciences, Falcon&#160;</td>
			<td dir="LTR">35 2070&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Petri dish (90 x 15 mm)&#160;</td>
			<td dir="LTR">Thermo Fisher Scientific&#160;&#160;</td>
			<td dir="LTR">BSN 101VR20&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">100 &#956;m cell strainer&#160;&#160;</td>
			<td dir="LTR">BD&#160;&#160;</td>
			<td dir="LTR">352360</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Peristaltic pump&#160;</td>
			<td dir="LTR">Watson Marlow&#160;</td>
			<td dir="LTR">SciQ 300&#160;&#160;</td>
			<td>Rinse tubing before and after each usage with sterile PBS and 70% ethanol.</td>
		</tr>
		<tr>
			<td dir="LTR">24-well plates</td>
			<td dir="LTR">Corning&#160;&#160;</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">96-well plates&#160;</td>
			<td dir="LTR">Corning&#160;&#160;</td>
			<td dir="LTR">3595</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Plate reader</td>
			<td>BMG Labtech</td>
			<td>FLUOstar Omega</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Serrefine forceps</td>
			<td>Hammacher GmbH&#160;</td>
			<td>Art. Nr. HSE 004-35 / Cat. Nr. 221-0051</td>
			<td>The serrefine forceps allow to clamp the vessel cannulated with the 23G needle without the need of holding the forceps during the perfusion procedure. URL: (http://www.hammacher.de/Laboratory-Products/Clamps-forceps/Serrefines/HSE-004-35-Serrefine::25126.html)&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Flow cytometry tubes</td>
			<td>BD Biosciences, Falcon&#160;</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Microcentrifuge tubes (1.5 ml)</td>
			<td>Elkay</td>
			<td>&#160; 000-MICR-150</td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Cell scraper</td>
			<td>BD Biosciences, Falcon</td>
			<td>353086</td>
			<td>Cut blade extremities with a pair of scissors to scrape cells in 24-well plates.</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td dir="LTR">Name of the Reagent</td>
			<td dir="LTR">Company&#160;</td>
			<td dir="LTR">Catalog number&#160;</td>
			<td dir="LTR">Comments/Description&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">&#160;EGTA (Ethylene Glycol Tetraacetic Acid)/HBSS (Hank&#39;s Balanced Salt Solution) Solution&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">HBSS containing 0.5 mM EGTA and 25 mM HEPES. Adjust pH to 7.4. Prepare 50 ml for each liver to perfuse.&#160;&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Collagenase Solution&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">DMEM low glucose containing collagenase type IV at 100 UI/ml, 15 mM HEPES and 1% Penicllin/Streptamycin (v/v). Adjust pH to 7.4. Prepare 100 ml for each liver to perfuse. After adding the collagenase, it is advised to warm up the solution for 30 min before use. This allows the collagenase activity to be optimum.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Kupffer Cell Isolation Medium&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">RPMI containing, 1% Non-Essential Amino-Acids (v/v),1% glutamax&#174; (v/v) and 1% Penicllin/Streptomycin (v/v). Prepare at least 100 ml for 1-3 livers.&#160;&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Kupffer Cell Culture Medium&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">RPMI containing 10% Fetal Bovine Serum, 1% Non-Essential Amino-Acids (v/v),1% Glutamax&#174; (v/v) and 1% Penicllin/Streptamycin (v/v). Prepare at least 100 ml for 1-3 livers.&#160;&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">SIP (solution of isotonic coated silica particles)</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">Mix 1.7 ml of 10x Phosphate Buffered Saline&#160; with 15.3 ml of Percoll&#174; to obtain 17mL of SIP.</td>
		</tr>
		<tr>
			<td dir="LTR">25% SIP solution&#160;</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">Mix 5 ml of SIP with 15 ml of 1x Phosphate Buffered Saline&#160;&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">50% SIP solution&#160;</td>
			<td dir="LTR"></td>
			<td></td>
			<td dir="LTR">Mix 10 ml of SIP with 10 ml of 1x Phosphate Buffered Saline&#160;</td>
		</tr>
		<tr>
			<td dir="LTR">Lysis Buffer</td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR">DMEM media with 0.9% Triton X-100</td>
		</tr>
		<tr>
			<td dir="LTR">PBS/BSA Solution</td>
			<td dir="LTR"></td>
			<td></td>
			<td dir="LTR">Prepare fresh Phosphate Buffered Saline pH 7.4 with 1% Bovine Serum Albumin.&#160;</td>
		</tr>
		<tr>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
			<td dir="LTR"></td>
		</tr>
	</tbody>
</table>

kupffer cell isolation for nanoparticle toxicity testing

الغالبية العظمى من الدراسات في المختبر nanotoxicological استخدمت خطوط الخلايا خلد عن التطبيق العملي لها. ومع ذلك، فقد أظهرت النتائج من جسيمات متناهية الصغر اختبار السمية في خطوط الخلايا خلد أو الخلايا الأولية التناقضات، مما يبرز الحاجة إلى توسيع استخدام الخلايا الأولية للفحوصات في المختبر. يصف هذا البروتوكول عزلة الضامة كبد الفأر، وخلايا كوبفر اسمه، واستخدامها لدراسة سمية جسيمات متناهية الصغر. خلايا كوبفر هي السكان بلعم الأكثر وفرة في الجسم، وتشكل جزءا من الجهاز الشبكي البطاني (RES)، المسؤولة عن القبض على تعميم النانوية. ويستند كوبفر طريقة عزل الخلايا ذكرت هنا على طريقة التروية 2-خطوة تليها تنقية على التدرج الكثافة. طريقة، بناء على الهضم كولاجيناز والكثافة الطرد المركزي، ومقتبسة من البروتوكول الأصلي الذي وضعته Smedsrød وآخرون. مصممة للفئران خلايا الكبد isolatioن ويقدم عالية الغلة (ما يصل إلى 14 × 10 6 خلايا لكل الماوس) ونقاء عالية (&gt; 95٪) من خلايا كوبفر. هذه الطريقة العزلة لا يتطلب معدات متطورة أو باهظة الثمن، وبالتالي يمثل حلا وسطا مثاليا بين التعقيد والعائد الخلية. استخدام الفئران أثقل (35-45 ز) يحسن العائد من طريقة العزل ولكن أيضا يسهل بشكل ملحوظ الإجراء من الوريد البابي إقناء؛ إدخال القنية. وقد تم قياس سمية أنابيب الكربون النانوية functionalized و -CNTs في هذا النموذج من قبل فحص LDH تعديلها. هذا الأسلوب يقيم بقاء الخلية عن طريق قياس انعدام السلامة الهيكلية للغشاء الخلية كوبفر بعد الحضانة مع -CNTs و. سمية الناجم عن -CNTs و يمكن قياسها باستمرار باستخدام هذا الاختبار، مشددا على أن خلايا كوبفر معزولة مفيدة لاختبار السمية جسيمات متناهية الصغر. الفهم الشامل للnanotoxicology يمكن أن تستفيد من هذه النماذج، مما يجعل اختيار جسيمات متناهية الصغر للترجمة السريرية أكثر ايفيcient.

وكان عدد الخلايا متني غير النقاء بما يتفق وتراوحت ما بين 8 و 14 × 10 6 خلايا في الماوس (تم تنفيذ 17 العزلة). وكانت كل العزلة الماوس كافية لوحة 16-28 الآبار. وأظهرت بقاء الخلية التي تلطيخ التريبان الأزرق بقاء الخلية ~ 95٪. أظهرت خلايا كوبفر شكل دائري غضون 30 دقيقة من الحضانة ...

Watch this Scientific Journal Video about Kupffer Cell Isolation for Nanoparticle Toxicity Testing at JoVE.com

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

كوبفر عزل الخلايا للجسيمات متناهية الصغر سمية اختبار

The large majority of in vitro nanotoxicological studies have used immortalized cell lines for their practicality. However, results from nanoparticle ...

kupffer-cell-isolation-for-nanoparticle-toxicity-testing

Research

JoVE Journal

Immunology and Infection

23.5K Views.  King's College London. Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells. 

Video: Kupffer Cell Isolation for Nanoparticle Toxicity Testing

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

عزل خلايا المناعة وظيفية القلب

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

عزل مجموعات فرعية B-الخلية السلائف من دم الحبل السري

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

الكشف عن جسيمات متناهية الصغر نيون التفاعل مع مجموعات سكانية فرعية خلية المناعة الأولية من قبل التدفق الخلوي

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

عزل الخلايا اللمفاوية من الجلد البشري لتحليل المظهري و<em&gt; فيفو السابقين</em&gt; زراعة الخلايا

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

التصوير كله والحيوان وتدفق Cytometric تقنيات CD8 + T الردود خلية التحليل من Antigen محددة بعد الجسيمات النانوية التطعيم

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

العزلة وتحليل تدفق Cytometric من الخلايا المناعية من الدماغ الدماغية ماوس

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

عزل وتوصيف والفحص الوظيفي لشبكة خلية اللثة المناعي

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

على طريقة العائد فعالة وعالية لعزل مجموعات فرعية خلية الفأر الجذعية

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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

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

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

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

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

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

أسلوب نانوحبيبات الفضي للتخفيف من حدة المتلازمة الكبدية الصفراوية في الفئران

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...