Bu eser Kanada Enstitüleri Sağlık Araştırma tarafından (148808 ve 148792) desteklenmiştir, SE bir kalp ve inme Vakfı, personel Ödülleri İstanbul İl Office, Mart Dimes, kalp araştırma ve Peter Munk Ted Rogers Merkezi tarafından desteklenmiştir Kalp Merkezi. XCC bir CIHR Banting bursu tutar. LA bir kalp ve inme/Richard Lewar öğrencilik Ödülü almıştır.

Etik Beyanı: Bu protokol gözden geçirilmiş ve Üniversitesi Sağlık Ağı (Toronto, Kanada), hayvan bakımı Komitesi tarafından onaylanmış ve hayvan bakımı Kanada Konseyi ile uyumlu.
1. arabellek hazırlık
<ol><li>HBB arabellek (Hank'in dengeli tuz çözüm (HBSS), % 2 ısı inaktive Sığır serum, %0,2 sığır serum albümin) hazırlayın. Isı 10 mL Sığır serum ve HBSS 500 ml Sığır serum albümin 1 g inaktive ekleyin. Filtre 0.2 µm filtreden sterilize ve 4 ° C'de depolayın</li>
	<li>FACS arabellek hazırlamak (fosfat tamponlu tuz (PBS), % 2 ısı inaktive Sığır serum, 1 mM Ethylenediaminetetraacetic asit (EDTA, pH 8.0)). Sığır serum ve 1 mL 0.5 M EDTA PBS 500 ml 10 mL ısı inaktive ekleyin. Filtre 0.2 µm filtreden sterilize ve 4 ° C'de depolayın Not: Çözümler 4 ° C'de 1 ay boyunca saklanır.</li>
</ol>2. lökosit dolaşan etiketleme
<ol><li>Aseptik steril fosfat tamponlu tuz (PBS) için 200 µL/fare son enjeksiyon hacmi içinde seyreltilmiş antikor 1 µg ile Anti-fare CD45 enjeksiyon hazırlayın. Birim 28,5 G insülin enjektör yük ve ışıktan uzak tutun. Not: iki farklı fluorochrome etiketli anti-CD45 antikorlar intrakardiyak boyama üzerinden intravasküler (damar yolu yönetim lekeli) ayırt etmek için kullanın (sindirim sonra lekeli; bkz. Adım 4).</li>
	<li>
Fareler (8-12 hafta yaşlı) kırmızı ısı lamba 5 min için kuyruğuna açarak önceden ısıtmak. Dikkat: hayvan aşırı değil. Lamba güvenli bir mesafede tutmak (> 30 cm).<ol>
<li>Hayvan sternal konumda uygun bir aygıt kullanarak engelleyin. Ortaya çıkarmak ve sigara dominant el ile kuyruk kuyruk dizin ve orta parmak ile altyapı ve kuyruk başparmak ve parmak yüzük ile orta-distal bölge immobilizing tarafından stabilize. Dikkat: cilt çıkardı gibi çok fazla güç kuyruk ucunda geçerli değildir. İğne eğimi damar içine yukarı dönük olacak şekilde yerleştirin. Gibi damar ponksiyon kolay iğne her zaman ven paralel tutun. Not: kan şırınga girerek oluşabilir ve iyi bir enjeksiyon işaretidir. İğne 90 ° açı bükme damardan yönetim kolaylaştırabilir. Bükme iğneler bir tehlike temsil edebilir gibi ile emanet ofis onaylayın.</li>
		<li>200 µL intravenöz kuyruk damar içine enjekte. Sıvı kolay akışını ve geçici olarak ven temizleyin. Dikkat: direnç karşılandığında sıvı zorlamayın. Bu iğne saklanmalıdır işaretidir.</li>
		<li>Antikor insanca fare ötenazi önce 5 min için dolaşmaya izin.</li>
	</ol>
</li>
</ol>3. kalp yalıtma ve sindirim
<ol><li>
CO2 kullanarak fare ötenazi veya diğer kurumsal olarak kabul edilen ötanazi yordam tarafından.
	<ol>
<li>CO2 ötenazi, CO2 odasında fareyi getirin ve dakikada (odası 10 L, 2 L/dk dolgu iseYani, ) Odası hacmi %20 için dolgu oranını ayarlamak. Nefes kesildiği tarih vardır kadar 2 dk ve monitör mouse için çalıştırın.</li>
		<li>CO2 kes ve ek bir 1-2 dk. nefes odası açmadan önce kesildiği tarih vardır sağlamak için fare ve fare fare işlemeye devam etmeden önce öldü onaylamak için bir ayak çimdik gerçekleştirmek.</li>
	</ol>
</li>
	<li>Sprey % 70 etanol ile fare. Karın, cilt kaldırın ve fare kadar göğüs kemiği kalça düzeyinde ventral orta hat başlangıç boyunca keserek incelemek. Karın açar ve diyafram ortaya çıkarmak için karaciğer gider.</li>
	<li>
Diyafram, sonra kaburga akciğer ve kalp delinme önlemek için dikkatli olmak orta çizgi, her iki tarafta kesti.
	<ol>
<li>Peeling ezbere geri kaburga, sonra Sol kulakçık sağ kulakçık tarafından takip kesim ilk maruz. Dikkat: Bu organlarda bulunan kan yıkama etkinliğini azaltabilir gibi herhangi bir arter veya damarlar yaralandı, sağlanması atria, kesme dikkatli kullanın.</li>
		<li>15-20 mL soğuk PBS 21 G iğne ile donatılmış bir 60 mL şırınga kullanarak ve soğuk PBS kalple sıvı. Yavaşça kalp Bankası forseps ile kavramak ve tepe yakınındaki sol ventrikül iğne yerleştirin. Sağ ventrikül yordamı yineleyin. Uygun bir enjeksiyon beyaz akciğerler ve soluk karaciğer ile sonuçlanır. Not: Perfüzyon ötenazi sonra 5 dakika içinde organ hücreleri dolaşan ile ilgi kontamine kan pıhtılaşması önlemek için yapılmalıdır.</li>
	</ol>
</li>
	<li>
Kalp Bankası forseps ile tutun ve hafifçe yukarı çekin kalp, sonra kalp ana pulmoner arterler ve damarlar, aort ve perikardiyal çuval keserek ayırmak.
	<ol>
<li>Kalp kalp başparmak ve işaret parmakları arasında bir temiz havsız kağıt havlu ile nazikçe tutarak temiz ve kulakçık ve ana arterler ve damarlar ve diğer kirletici maddeleri kalıntıları forseps ile çekin. Not: kulakçık tamamı kaldırılır ve bu dokularda kendi yerleşik bağışıklık hücreleri gibi hiçbir diğer dokular, akciğer veya kan damarları, gibi mevcut olun. Akciğer özellikle bu Miyokardiyum içinde daha küçük nüfus maske yapabilirsiniz bir büyük bağışıklık hücre nüfusa sahiptir.</li>
	</ol>
</li>
	<li>
Kalp 50 µL soğuk PBS ile 5 cm plastik çanak yerleştirin.
	<ol>
<li>Görünür hiçbir parça vardır ve pürüzsüz bir tutarlılık vardır kadar eğri diseksiyon makası kullanarak kalp kıyma. Not: kalp doku kaybı en aza indirmek için kesme sırasında yemeğin bulunduğu bir alanda bulundurmaya çalışın.</li>
		<li>2 mL microcentrifuge tüp ile 800 µL soğuk Dulbecco'nın modifiye kartal Orta (DMEM) içine eğri forseps kullanarak thetrituratedheart doku aktarın.</li>
	</ol>
</li>
	<li>
Ben, hyaluronidase tip ı-S 60 U/mL ve DNaz 60 U/mL 450 U/mL ofcollagenase ekleyin-ben her örnek için.
	<ol>
<li>45-60 dk 50 RPM sallanan bir shaker olarak 37 ° C'de örnekleri kuluçkaya.</li>
	</ol>
</li>
	<li>
Sindirim, girdap örnekleri kısa bir süre sonra (20 s) ve hemen buz örnekleri 4 ° C'de tutmak için yer
	<ol>
<li>Doku örnekleri homojen ve Pipet Ucu zarar değil kadar 1 mL pipettor kullanarak, örnekleri yukarı ve aşağı pipet. Not: bir kaç kez pipet için tanımlama tekrarlanabilirlik artabilir. Kalpleri en iyi şekilde sindirmek değil eğer Miyokardiyum adet pipet engel. Yavaşça microcentrifuge tüp karşı pipet ucu Bileme daha büyük doku parçaları homojenize yardımcı olacaktır.</li>
	</ol>
</li>
	<li>
50 mL konik tüp ile 1 mL soğuk HBB yerleştirilen bir 40 µm hücre süzgeç önceden ıslak.
	<ol>
<li>Homojenize örnek için filtre ekleyin ve yavaş yavaş 12-14 mL soğuk HBB dökerek ile yıkayın.</li>
		<li>Filtre uygulanmış örnekleri etiketli 15 mL konik tüp aktarmak ve 4 ° C'de tutmak</li>
	</ol>
</li>
	<li>
Santrifüj örnekleri 400 x g 4 ° C'de 5 min için de Süpernatant Aspire edin.
	<ol>
<li>Her örnek için 1 mL amonyum klorür potasyum (ACK) lizis arabelleği ekleyerek kırmızı kan hücreleri parçalayıcı. Örnekleri vortexing tarafından resuspend ve 5 min için oda sıcaklığında kuluçkaya.</li>
		<li>Lizis tamamladıktan sonra 5-8 mL, soğuk Hank'in dengeli tuz çözüm (hemoliz durdurmak için HBSS) ekleyin.</li>
	</ol>
</li>
	<li>Örnekleri, 400 x gfor 4 ° C'de 5 dk santrifüj kapasitesi Süpernatant Aspire edin.</li>
	<li>Her örnek ve etiketli 1.5 mL microcentrifuge tüp transferi FACS arabelleği 1 mL ekleyin. Not: Protokol burada en fazla 2 saat için 4 ° C'de depolanan örnekleri ile duraklatılmış</li>
</ol>4. akış sitometresi boyama ve analiz
<ol><li>
Santrifüj örnekleri 400 x g 4 ° C'de 5 min için de
	<ol>
<li>Non-spesifik antikor bağlama azaltmak için seyreltik 1: 100 anti-CD16/CD32 ve monosit engelleyici (1:20) (bakınız Tablo reçetesi) muhasebe örnek başına 50 µL FACS için. 15 dakika oda sıcaklığında kuluçkaya.</li>
		<li>Antikor seyreltme (1:250 her antikor için) muhasebe FACS 50 µL örnek başına için hazırlayın. Not: Bir canlılık boya antikor kokteyl ölü hücreleri dışlamak için eklenebilir. Alternatif olarak, küçük boyutlu dışlama akışı analizi tarafından kullanılan (Şekil 1C ve D) olabilir.</li>
		<li>Çamaşır olmadan, 50 µL antikor ana karışımı her örnek ve karışımı ekleyin.</li>
		<li>Örnekleri için karanlıkta 30 dk 4 ° C'de kuluçkaya.</li>
	</ol>
</li>
	<li>Yıkama ve örnekleri, 400 x g 4 ° C'de 5 dakika santrifüj kapasitesi için her örnek 600 µL FACS eklemek</li>
	<li>Süpernatant Aspire edin ve Pelet 300 µL FACS içinde resuspend. Karanlıkta 4 ° C'de örnekleri tutmak. Not: Bazı canlılığı boya, DAPI gibi akış sitometresi çalıştırmadan önce 10 dakika içinde boyama gerektirir.</li>
	<li>Akış Sitometresi tarafından analiz (bkz: Şekil 1 ve 2) sonraki 3 saat içinde. Eğer daha uzun bir süre düşük konsantrasyon paraformaldehyde kullanarak gerekli, kısa vadeli fiksasyon (0.5-%1 PBS) boyama korumak için önerilir. Not: Akış sitometresi akışkanlar tıkanıklığı önlemek için bir 40 µm hücre süzgeç önce başlatmak Akış Sitometresi Analizi yoluyla örnekleri filtre uygulamak için önerilir.</li>
</ol>

<ol>
	<li>Marboe, C. C., Fenoglio, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+and+natural+history+of+human+myocarditis.">Pathology and natural history of human myocarditis.</a> Pathology and Immunopathology Research. 7 (4), 226-239 (1988).</li><li>Epelman, S., 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=Embryonic+and+adult-derived+resident+cardiac+macrophages+are+maintained+through+distinct+mechanisms+at+steady+state+and+during+inflammation.">Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.</a> Immunity. 40 (1), 91-104 (2014).</li><li>Pinto, A. R., 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=An+abundant+tissue+macrophage+population+in+the+adult+murine+heart+with+a+distinct+alternatively-activated+macrophage+profile.">An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.</a> PLoS One. 7 (5), 36814 (2012).</li><li>Lavine, K. J., 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=Distinct+macrophage+lineages+contribute+to+disparate+patterns+of+cardiac+recovery+and+remodeling+in+the+neonatal+and+adult+heart.">Distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16029-16034 (2014).</li><li>Fujiu, K., Wang, J., Nagai, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardioprotective+function+of+cardiac+macrophages.">Cardioprotective function of cardiac macrophages.</a> Cardiovascular Research. 102 (2), 232-239 (2014).</li><li>Hulsmans, M., 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=Macrophages+Facilitate+Electrical+Conduction+in+the+Heart.">Macrophages Facilitate Electrical Conduction in the Heart.</a> Cell. 169 (3), 510-522 (2017).</li><li>Nahrendorf, M., 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=The+healing+myocardium+sequentially+mobilizes+two+monocyte+subsets+with+divergent+and+complementary+functions.">The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions.</a> Journal of Experimental Medicine. 204 (12), 3037-3047 (2007).</li><li>Clemente-Casares, X., 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=A+CD103(+)+Conventional+Dendritic+Cell+Surveillance+System+Prevents+Development+of+Overt+Heart+Failure+during+Subclinical+Viral+Myocarditis.">A CD103(+) Conventional Dendritic Cell Surveillance System Prevents Development of Overt Heart Failure during Subclinical Viral Myocarditis.</a> Immunity. 47 (5), 974-989 (2017).</li><li>Van der Borght, K., 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=Myocardial+Infarction+Primes+Autoreactive+T+Cells+through+Activation+of+Dendritic+Cells.">Myocardial Infarction Primes Autoreactive T Cells through Activation of Dendritic Cells.</a> Cell Reports. 18 (12), 3005-3017 (2017).</li><li>Galkina, E., 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=Preferential+migration+of+effector+CD8++T+cells+into+the+interstitium+of+the+normal+lung.">Preferential migration of effector CD8+ T cells into the interstitium of the normal lung.</a> Journal of Clinical Investigation. 115 (12), 3473-3483 (2005).</li><li>Edelson, B. T., 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=Peripheral+CD103++dendritic+cells+form+a+unified+subset+developmentally+related+to+CD8alpha++conventional+dendritic+cells.">Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8alpha+ conventional dendritic cells.</a> Journal of Experimental Medicine. 207 (4), 823-836 (2010).</li></ol>

Yazarlar ifşa gerek yok.

Miyokardiyal iltihap veya miyokardit, en kalp-damar hastalıkları bir özelliktir. Ancak, Miyokardiyum sigara hastalık durumlarında kendi bağışıklık bileşenlerinde yoksun değildir. Kararlı duruma sırasında birçok bağışıklık hücreleri Miyokardiyum içinde bulunan ve bakım ve koruma önemli rol oynarlar. Bu farklı nüfus hücrelerin karakterizasyonu bu protokol için sunulan bir gibi yöntemleri olmadan mümkün olmazdı.
Miyokardiyum mekanik ve enzimatik sindirim bir arada...

Miyokardiyal stres veya yaralanma, iskemik dahil olmak üzere (iskemi reperfüzyon veya miyokard infarktüsü) farklı formları ve iskemik olmayan (hipertansiyon veya miyokardit), ama aynı zamanda Onarıcı ve koruyucu, inflamatuar hücrelerle istihdamı teşvik patojenik özellikleri. 1891, gibi erken Romberg ilk hücresel infiltratlar tifüs ve Kızıl hastalığı1ile infekte hastaların Miyokardiyum varlığı tarif. Ancak, kardiyak bağışıklık hücreleri detaylı çalışmanın daha gelişmiş immunophenotyping teknikleri geliştirilmesi gerekmektedir. Sonuç olarak, yakın zamanda bağışıklık hücreleri temel bakım rolleri ile farklı bir popülasyon sırasında kararlı duruma Miyokardiyum içinde bulunduğu anlamaya başlamıştır.
Histoloji oldu ve hala, enflamasyon sırasında kalp bağışıklık hücreleri ayırdetmek için en yaygın yöntem. Ancak, Histoloji değerli bir tanı aracı temsil ederken, kullanımı kardiyak bağışıklık hücreleri insan önemli sınırlamaları vardır. Miyokardiyum içinde ikamet eden bağışıklık hücreleri toplam hücrelerin çok küçük bir oran temsil ve orantılı olarak çok büyük bir alanda daha fazla veya daha az eşit olarak dağıtılır. Benzer şekilde, kardiyak inflamasyon, viral miyokardit gibi çeşitli formları inflamasyon odak desenleri gösterin. Kalp bağışıklık sisteminin doğru analiz kez yüksek derece histolojik örnekleme, önyargı azaltmak için maliyeti artan gerektiren anlamına gelir. Ayrıca, Histoloji çok sınırlı bir hücre kimliği (Örneğin sayıda parametre) kullanışlı bilgileri sağlar. Akış Sitometresi (yüzey işaretleyicileri, transkripsiyon faktörleri veya salgılanan moleküller gibi) birden çok ifade işaretleri kullanımını gerektiren hücre alt kümeleri artan bir sayı ile immunophenotyping için en güçlü araç olarak kendisini kurmuştur, düşük maliyetli ve yüksek işlem hacmi nedeniyle.
İmmunophenotyping teknikleri çok sayıda hücreleri tek hücre analizi için izin verimli sindirim protokolleri gelişimini yakından bağlıdır. Hücre ekstraksiyon ayrıntılı iletişim kuralları gelişimi kardiyak İmmünoloji çalışmada fırsatları yeni bir pencere açtı. Arada sindirim, akış sitometresi ve transcriptomics yoluyla, makrofajlar ve dendritik hücreleri merkezinde ikamet eden büyük nüfus karakterize2,3olmuştur. Kardiyak bağışıklık hücreleri en bol popülasyon sırasında kararlı duruma vardır CD64+MerTK+ CCR2 veya CD11c2onların ifade üzerinde temel alan makrofajlar, daha da ayrılabilir. CCR2+ ise makrofajlar yetişkin kemik iliği hematopoiesis kaynaklanan çoğu CCR2 MHC-II yüksek veya düşük düzeyde ifade edebilir,- makrofajlar, esas olarak Doğum öncesi kökenlidir. Makrofajlar onarım4 ve enkaz5 kaldırılması sırasında yaralanma veya elektrik bağlantısı6 sabit durumda destekleyen gibi temel fonksiyonları yerine getirir. Enflamasyon sırasında çeşitleri Ly6CMerhaba MHC-IIlo CD64int monosit önemli bir akını oluşur, hangi daha sonra Ly6CMerhaba CCR2 ayırt+ makrofajlar2,7 . Önemli ölçüde daha küçük ancak önemli Vaha'da hücrelerinin sağlıklı bireyler Miyokardiyum içinde ikamet eden dendritik hücreler8,9/ oluşur. Kardiyak geleneksel DCs (KGK) iki ana alt kümeleri son zamanlarda nitelendirmiştir: cDC1 (CD103+ DCs) ve cDC2 (CD11b+ DCs). DCs enfeksiyon8 karşı savunmak için önemli rol oynarlar ama aynı zamanda kendini yaralama sırasında özellikle9miyokard infarktüsü enflamasyon sırasında yükseltebilirsiniz.
Burada, fare Miyokardiyum üzerinden kardiyak immün hücrelerin izolasyonu için basit bir yöntem açıklanmaktadır. Yöntemi analiz veya daha fazla akış sitometresi sıralama veya manyetik boncuk zenginleştirme tarafından saf bir tek hücre süspansiyon elde etmek için enzimatik ve mekanik sindirim bir hücre-Süzgeç filtrasyon ile birleştirir. Extravascular miyokard hücrelerinin doğru ölçümler kardiyak perfüzyon kardiyak microvasculature kan olası kirletici kaldırmak için gerektirir. Buna ek olarak, biz daha fazla intravasküler kirleticiler, Galkina ve ark. Protokolü10tabanlı miyokardiyal hücreler ayırt etmek için kullanılan bağışıklık hücrelerinin damar içi etiketleme, isteğe bağlı bir adım mevcut. Son olarak, biz büyük makrofaj ve cDC altgrupları tanımlamak için bir temel Akış Sitometresi Analizi mevcut.

<table border="0" cellpadding="0" cellspacing="0" style="width:815px;" width="814">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Phosphate buffered saline</td>
			<td style="width:123px;">Wisent</td>
			<td style="width:185px;">311-010-CL</td>
			<td style="width:167px;">1x PBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">21Gx 1 1/2 (0.8mmx40mm) PrecisionGlide Needle</td>
			<td>BD</td>
			<td>305167</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hank&#8217;s Balanced Salt Solution</td>
			<td>Wisent</td>
			<td>311-511-CL</td>
			<td>1x HBSS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A4503-50G</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bovine serum</td>
			<td>Sigma</td>
			<td>B9433</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0.5M Ethylenediaminetetraacetic acid</td>
			<td>BioShop</td>
			<td>EDT111</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vacuum filter , 0.2 &#181;m Filtropur V50</td>
			<td>Sarstedt</td>
			<td>83.1823.001</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">28G 1/2 1 cc insulin syringe</td>
			<td>BD</td>
			<td>329424</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">60 mL syringe</td>
			<td>BD</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Wisent</td>
			<td>319-005-CL</td>
			<td>1x DMEM with 4.5 g/L glucose and L-Glutamine and Sodium Pyruvate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Collagenase I</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>from Clostridium histolyticum</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hyaluronidase type I-S</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNase-I</td>
			<td>Sigma</td>
			<td>D4513</td>
			<td>from bovine pancreas</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell strainer, 40 &#181;m Nylon</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ammonium-Chloride- Potassium (ACK) lysis buffer</td>
			<td>Lonza</td>
			<td>10-546E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa Fluor 700 anti-mouse/human CD11b</td>
			<td>Biolegend</td>
			<td>101222</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC/Cy7 anti-mouse Ly-6c</td>
			<td>Biolegend</td>
			<td>128025</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">APC anti-mouse CD103</td>
			<td>Biolegend</td>
			<td>121414</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Brilliant Violet 605 anti-mouse CD11c</td>
			<td>Biolegend</td>
			<td>117334</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE anti-mouse Ly-6G</td>
			<td>Biolegend</td>
			<td>127607</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pacific Blue anti-mouse I-Ab</td>
			<td>Biolegend</td>
			<td>116422</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC anti-mouse CD64 (FcgRI)</td>
			<td>Biolegend</td>
			<td>139315</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE/Cy7 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103113</td>
			<td>1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP/Cy5.5 anti-mouse CD45</td>
			<td>Biolegend</td>
			<td>103132</td>
			<td>1:40 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TruStain fcX (anti-mouse CD16/32)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td>1:100 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">True-Stain Monocyte Blocker</td>
			<td>Biolegend</td>
			<td>426101</td>
			<td>1:20 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo V10</td>
			<td>TreeStar Inc</td>
			<td></td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse: Batf3-/-</td>
			<td>The Jackson Laboratory</td>
			<td>JAX: 013755</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and identification of extravascular immune cells of the heart

Bağışıklık sistemini sağlıklı bir kalp, temel bir bileşenidir. Miyokardiyum olduğunu farklı bağışıklık hücre alt kümeleri fonksiyonel bölünebilme kararlı duruma sırasında ve çeşitleri enflamasyon sırasında ile zengin bir nüfusa ev. Yakın zamana kadar kalbinde bağışıklık hücreleri çalışma mikroskobu kullanımını gerekli veya yeterli duyarlılığı sırasında şiddetli inflamasyon sağlanan ama güvenle aciz kalmışlardır kötü gelişmiş sindirim protokollerini tanımlamak küçük — ama anahtar — nüfus hücreleri kararlı duruma sırasında. Burada, bir basit yöntem birleştirerek enzimatik (collagenase, hyaluronidase ve DNaz) ve küçük ayırt etmek için antikorlar fluorescently etiketli intravasküler yönetimi tarafından öncesinde fare kalplerin mekanik sindirim tartışmak ama kaçınılmaz damar içi hücre kirletici. Bu yöntem tarafından incelendi akış sitometresi tanıma, fenotipleme ve miktar veya floresan aktif hücre sıralama veya manyetik boncuk ayrılması için daha fazla saf izole hücrelerin bir süspansiyon oluşturur Transkripsiyon analiz veya vitro çalışmalar. Biz anahtar makrofaj ve dendritik hücre popülasyonlarının kalp ayırt etmek için bir adım adım akış sitometrik analizin bir örnek içerir. Bir orta ölçekli deney (10 kupa) yordamı tamamlandıktan 2 – 3 saat gerektirir.

Bugüne kadar iyi bir yöntem yoktur cardiomyocytes gibi diğer kardiyak hücre bileşenlerinden bağışıklık hücreleri izole etmek için tasarlanmıştır. Bu nedenle, kardiyak tek hücre süspansiyon tarafından Akış Sitometresi Analizi bir tek hücre ve küçük boyutlu dışlama gating (Şekil 1A) ardından bağışıklık hücre popülasyonlarının tanımlamak için CD45 ile önceden geçişi gerektirir. Alternatif olarak, ölü h...

Watch this Scientific Journal Video about Isolation and Identification of Extravascular Immune Cells of the Heart at JoVE.com

Isolation and Identification of Extravascular Immune Cells of the Heart

Yalıtım ve kalp Extravascular bağışıklık hücreleri tanımlaması

Bu iletişim kuralı izole, tanımlamak ve kararlı duruma veya enflamasyon sırasında fareler Miyokardiyum içinde ikamet eden bağışıklık hücreleri ölçmek için basit ve etkili bir yöntem sunuyor. Protokol enzimatik ve mekanik sindirim daha fazla akış sitometresi tarafından analiz edilecek bir tek hücre süspansiyon nesil için birleştirir.

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

isolation-identification-extravascular-immune-cells

Research

JoVE Journal

Immunology and Infection

12.4K Görüntüleme.  University Health Network (UHN). Bu iletişim kuralı izole, tanımlamak ve kararlı duruma veya enflamasyon sırasında fareler Miyokardiyum içinde ikamet eden bağışıklık hücreleri ölçmek için basit ve etkili bir yöntem sunuyor. Protokol enzimatik ve mekanik sindirim daha fazla akış sitometresi tarafından analiz edilecek bir tek hücre süspansiyon nesil için birleştirir.

Video: Isolation and Identification of Extravascular Immune Cells of the Heart

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Sabit ve Yaşam Hücreler Sitometrisi ile Calpain Aktivitesi Ölçme

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

İmmünoloji ve Enfeksiyon

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hücreler stellat Yedi Adımda

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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.

Fonksiyonel Kardiyak Bağışıklık Hücreleri İzolasyon

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 ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Paraziter Eşek Arıları Bir Giriş<em&gt; Drosophila</em&gt; Ve antiparaziter İmmün Yanıt

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

2-Foton Mikroskopi kullanarak Fare Thymus intravital Görüntüleme

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Kemik İliği Transplantasyonu tarafından Fare Kolit immün ve non-immün Hücreler Gen Ekspresyonu İşlevsel Rolleri Farklılaşan

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Fonksiyonel ve Moleküler Çalışmalar Birincil murin Tip II alveol epitel hücrelerinde akım sitometri İzolasyonu

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

Yağ Doku Bağışıklık Hücre İzolasyonu

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

İzolasyon, tanımlama ve Murin Thymic Epitel Hücreleri saflaştırılması

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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.

İskemik Fare Beyin gelen Bağışıklık Hücreleri İzolasyon ve Akım Sitometrisi Analizi

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