This work is supported by a Research Grant from Dialysis Clinics Inc. and from the University of Missouri Research Board Grant.

gerçekleştirilen tüm protokol adımları gözden ve Missouri Hayvan Bakımı ve Kullanımı Komitesi Üniversitesi (ACUC) tarafından onaylanmıştır. herhangi bir yaşta herhangi bir teorik kemirgen deneyleri için de kullanılabilir, ancak bu protokol için, 15 haftalık, erkek C57BL / 6 fareleri kullanılmıştır. Bu bir hayatta olmayan cerrahi olduğu için, ötenazi kan kaybından ve bilateral pnömotoraks ile sağlanır. Böbrekler 1. perfüzyon Not: kalp, karaciğer ve böbrek gibi organların Perfüzyon verilerin yorumlanması ile engel olabilir kanı temizler. Mümkünse Bu nedenle, biz her zaman organlar serpmek. <ol><li> o hareketsiz kadar (3 ml / dak) indüksiyon odası, bir izofluran fare yerleştirin. Sonra, odanın dışında fare kaldırmak diseksiyon masaya dorsal yattı ve fare burun üzerinde izofluran koni yerleştirin ve regülatör ile 1.5 ml / dk hızında izofluran itin. </li><li> ayak tutam tepk kontrolhayvan anestezi cerrahi uçağı elde etti ise cımbız ile e değerlendirmek. tuzlu su ile ventral karın temizleyin ve makas ile karın kesip açmak ve farenin sol tarafına (cerrahi alanı dışında kalan) karın organları itin. </li><li> ile inferior vena kava kan toplayın 25 gauge 1 cc şırınga iğne sabit ve 0.5M EDTA 10 ul içeren bir 1.5 ml mikrosantrifüj tüp ekleyin. Not: Kan Toplama isteğe bağlıdır. Plazma biyokimyasal ölçümler için de kullanılabilir. Lökositler akış sitometri deneyleri için kullanılabilmektedir. </li><li> makas kullanarak, sağ atriyum küçük bir kesim sonraki adımda kan ve PBS karışımı izin yapmak. </li><li> 50 cc şırınga alın ve buz soğukluğunda PBS (20-30 cc) ile doldurun. Sonra, bir şırınga için 21-25 G iğne takılır (forseps ile yavaşça kalp tutarken) tepe noktası, sol ventrikül delinerek yavaş boyunca fosfat tamponlu tuz, pH 7.4 (PBS) ile sol ventrikül serpmek1-2 dk. Not: Kalp hemen perfüzyon ilk birkaç mililitre üzerine blanches. Bu perfüzyon vena kava yoluyla venöz dönüş yoluyla karaciğer kanı drene olduğunu gösterir gibi, haşlama için karaciğer gözlemleyin. Buna ek olarak, bu sağ ventrikül patlağı gösterir gibi akciğer infüzyon sırasında genişleyen değil emin olun. akciğerler genişletmek için olsaydı, biraz geri sol ventrikül içine iğne çekilme ve perfüzyon devam edin. elde edilir ötanazi kadar ilk kesi alınan zaman az 2 dk </li></ol> Böbrekler 2. Diseksiyon <ol><li> perfüzyon tamamlandıktan sonra, ekleri (kan damarları, yağ ve adrenal bezler) ve tüketim sağ böbrek incelemek için makas ve forseps kullanabilir. </li><li> Yavaşça kesme ve forseps ile diseksiyon ile böbrek kaplayan kapsülü kaldırın. böbrek tartılır. Not: böbrek Tartı isteğe bağlıdır. Bununla birlikte, bazı deneylerBöbrek boyutu / doku gramı başına hücre verimi normalize etmek için kullanılabilmektedir ağırlık değişikliği ile sonuçlanabilir. </li><li> böbrek Tartıldıktan sonra,% 0.5 FBS ihtiva eden RPMI-1640 içinde 1 ml koyun veya lekelenme tampon çözeltisi sitometrisi (50 ml konik vidalı kapaklı tüp) akar ve daha sonra kullanılmak kadar buz üzerinde tüp yerleştirin. Not: açıklaması kolaylığı, biz boyama tampon çözelti flow sitometri bağışıklık hücrelerinin izolasyonu kalan anlatacağız için. Her iki böbrek veya böbrek 1 + 1/3 Rd (isteğe bağlı) alt deneylere bağlı olarak kullanılabilmesine rağmen ek olarak, tek bir böbrekten bağışıklık hücrelerinin izolasyonu anlatacağız. Böbrek 1/3 üçüncü protein / RNA deneyler için dondurulmuş olabilir ve 1/3 rd immünohistokimya veya transmisyon elektron mikroskobu için tamponlar içinde yerleştirilebilir. </li></ol> Böbrekler 3. Homojenizasyon <ol><li> örnek tanımlama bilgileri içeren bir 5-80 ml kapasiteli homojenizasyon çanta Etiket,iç süzgeci (STR) kaldırmak ve atın. buz Sitometrisi lekeleme tampon maddesi çözeltisi, 5 ml torbayı doldurmak tampona böbrek aktarın. İkinci numune veya işleme ihtiyaç daha fazla örnek için bu işlemi tekrarlayın. </li><li> bir yarım Akış Sitometri lekeleme tampon maddesi çözeltisi içine daldırılmış böbrek, diğer yarısı ise sadece üzerine katlanır, böylece, devre homojenleştirme torba katlayın. </li><li> Doku homojenleştirici açın ve hızlı (ayar) rpm olarak ayarlayın. alt kenarına yakın raket karşı karşıya böbrek katlanmış homojenizasyon çantası yerleştirin ama çanta alt uç kürekler alt sınırının altında kayan olmadığından emin olun. <ol><li> 2 kürek olduğu gibi aynı zamanda ikinci örnek işleyin. 120 sn olarak süreyi seçin. kürekler böbrekler homojenize için ileri ve geri hareket gözlemleyin. Not: Bu arada, bu kesinti ilk iki işlem sırasında örnekleri 2 fazla böbrek (veya başka doku) hazırlamak için kullanılabilir. </li></ol&gt; </li><li> homojenizasyon birinci seti ile tamamladıktan sonra, buz torbaları aktarmak ve böylece bir sonraki numune seti ve doku homojenleştirici yükleyin. Görme böbrekler yeterince homojenize ve hiçbir büyük parçalar görünür olduğundan emin olun. Not: Genellikle, hızlı rpm&#39;de doku homojenleştiricisi içinde 2 dakika böbrek için yeterlidir. </li><li> buz üzerinde 50 ml konik tüp içine 100 mikron hücre süzgeç yerleştirin. Sonra, hücre süzgecinden tamamen üzerinde Homojenat pipetle. 4 ° C de 1 dakika 50 xg bir taban santrifüj ayarlamak ve santrifüj örnekleri yükleyin. Not: Santrifüj Tüm homojenat (ihtiva eden hücreler) bir hücre süzgecinden geçirildi olduğunu garanti eder. </li><li> santrifüj tüpleri çıkarın ve buz üzerinde geri koyun. Hücre süzgeçler atın ve Homojenat tutun. eşit miktarda ekleyin (~ 6 mi) üniform bir hücre süspansiyonu yapmak için hücre peleti / yüzer tabakanın ve pipet ve aşağı yukarı birkaç kez 1 x RBC parçalama tamponu. visuaLly hemen süspansiyon azalma kırmızı rengin bazı gözlemleyin. Maksimum RBC parçalama elde etmek için 5 dakika boyunca buz üzerinde tüpler bırakın. Not: Araştırmacı böbrek perfüzyon ve daha az hücre manipülasyonu istenen emindir ise RBC parçalama adım atlanabilir. Biz RBC lizis hücre süspansiyonu tabi değil tarafından böbrek hücre veriminde önemli bir artış gözlemledik. </li><li> 5 dakika bir hücre tanesinin oluşturulması için 500 x g&#39;de, 4 ° C olarak ayarlandı santrifüj tüpleri dönerler. Adımlar 3.6 ve 3.7 sırasında koloidal silika (Percoll) tabanlı yoğunluk gradyan santrifüj hazırlamak Not:. <ol><li> alt polipropilen tüpler yuvarlak 10 ml alın ve ilgili örnek numaraları ile onları etiketlemek ve bir tüp rafa koyun. Not: Aşağıdaki 4 böbrek örnekleri için yeterli yoğunluk gradyan reaktifler yapmak açıklamaktadır. </li><li> Sitometrisi Boyama Tampon çözeltisi 7 ml alın ve bir 15 ml konik tüp içine pipetle. 2 M sükroz, 1 ml ilave edilir0.25 M sukroz hazırlar. </li><li> Sonra, başka bir 15 ml konik tüp içine yoğunluk gradyan reaktif ve pipet bunu 7.2 ml alır. % 72 yoğunluk gradyan reaktif ve doğrudan 0.25 M sükroz hazırlamak Sitometrisi lekeleme tampon maddesi çözeltisi 1.55 mi, 2 M sukroz, 1.25 ml ilave edilir. </li><li> Pipet her bir örnek için bir yuvarlak dipli polipropilen tüpe% 72 yoğunluk gradyan ayıracı 1 ml. </li></ol></li></ol> 4. dereceli santrifüj <ol><li> Adım 3.7 sonra tüpleri almak ve yavaşça tek bir hareketle yavaşça süpernatant süzün ve ayrıca tüp baş aşağı tutulurken oluşturan sıvının bir sonraki damla süzün. Not: Bu adım verimleri varyasyon oluşturmak için yeterli yoğunluk gradiyenti değiştirebilirsiniz aşırı sıvı geride bırakarak beri önemlidir. Tüp dik geri ayarlandığında, ~ sıvı 200-300 ul hücre pelet ile kalır. </li><li> Pipet ve yavaşça ama kuvvetlice hücre pelet üzerine 0.25 M sükroz 1 ml yukarı pipet ve Brin aşağıg muntazam süspansiyonuna hücreleri. Sonra,% 36 yoğunluk gradyan maddesi oluşturmak için hücreleri karıştırmak için şiddetle pipetle bir kez daha 1 ml% 72 yoğunluk gradyan reaktifi (3.7.4) hücre süspansiyonu ekleyin ve. </li><li> polipropilen Pasteur (transfer) pipet içine% 72 yoğunluk gradyan reaktif 1 ml alın. ampul bile basınç, yavaşça hücre süspansiyonu altına Pasteur pipeti (% 36 yoğunluk gradyan reaktif) eklemek ve bir oluşturmak için% 72 yoğunluk gradyan reaktifi boşaltın (ucunda sıvı sütunu tutar ve kabarcık oluşumunu engeller) farklı ağır tabaka. </li><li> yavaşça numune tüpleri almak ve bu aşama için, oda sıcaklığında bir santrifüj içine yerleştirin. 500 x g&#39;de 20-30 dakika süreyle Spin. </li><li> Bu arada, gelen örnek tanıtıcı ile alt santrifüj tüpleri yuvarlak polipropilen başka iki takım hazırlamak. </li><li> Adım 4.4 tamamlandıktan sonra, en az çalkalanarak yavaşça polipropilen tüpler içeren örneği kaldırmak ve aşağı ayarlayınTüp raf. </li><li> İlk örnek tüp ve ilgili boş tüp Pick up. Bir polipropilen Pasteur pipeti ile nazikçe ama iyice ölçülen emme ile üst &quot;önemsiz katmanı&quot; veya &quot;yapışkan malzeme&quot; kaldırmak ve boş yuvarlak dipli santrifüj tüpüne koyun. <ol><li> (Kırmızımtırak bir renk [kırmızı kan hücreleri] off-beyaz görünebilir) ulaşıldığında &quot;buffy coat&quot; kadar degrade üst kısmını kaldırarak devam edin. ~ 3 mm yükseklik &quot;buffy coat&quot; ulaşılır ise emiş durdurun. Not: Bu malzeme tamamen emme devam etmeden önce degrade üst kısmından gitti olduğundan emin olun. </li></ol></li><li> taze Pasteur pipet ve taze bir yuvarlak dipli santrifüj tüpü alın ve yavaşça buffy coat çıkarın. Hücrelerin en toplanmış ve uygun numune tanımlayıcı ile 2. yeni yuvarlak dipli tüpe eklenir sağlamak için, buffy coat altında 5 mm - 3 emme devam ediyor. </li><li> 1-2 ml o Eklef Sitometrisi Boyama Tampon 2. tüp çözüm ve yukarı ve aşağı pipetleme ile iyice karıştırın. </li><li> 4 ° C&#39;de 5 dakika boyunca 500 xg&#39;de hücreleri dönerler. Sitometrisi Boyama Tampon çözeltisi ile 2. yıkama adım isteğe bağlıdır. Sitometrisi Boyama Tampon çözeltisinin 500 ul ve yeniden süspanse hemasitometre kullanarak hücreleri saymak geçin. </li></ol> 5. İsteğe bağlı (Hücre Etiketleme) <ol><li> hücrelerin aynı sayıda, her numune Aliqout. 10 6 hücre başına 0.5 ug (anti-CD16 / CD32 FcR) bir seyreltme bloke antikor ilave edin ve 15 dakika boyunca buz üzerinde inkübe edilir. </li><li> önce gerçek deneyler, araştırmacı tarafından birbirleri ile iyi çalışması için belirlenir (uygun fluorophores konjuge) primer antikorlar istenen kokteyl ekleyin. 30 dakika boyunca buz üzerinde inkübe edin. Hücrelerin PBS ile yıkayın. Not: Bu yazının böbrek bağışıklık hücre etiketleme gösteri için, T-ly kullanılanmphocyte ve makrofaj / dendritik hücre paneli. BV421, Anti-Foxp3 (Clone: ​​FJK-16&#39;lar) APC, Anti-CD127 (Klon: A7R34): T-lenfosit paneli tamir edilebilir Canlılık Leke FVS510, anti-CD45 (30-F11 klonu) oluşuyordu PE / Cy7, anti CD44 (Klon: IM7) PerCP / Cy5.5, anti-CD4 (klon: RM4-5) APC-Cy7, anti-CD8 (Klon: 53-6,7) BV785. BV421, Anti-Ly6G (Klon: 1A8) FITC, anti-CD11 b (Klon: M1 / ​​70) PerCP-Cy5.5, Anti: makrofaj paneli tamir edilebilir Canlılık Leke FVS510, anti-CD45 (30-F11 klonu) oluşuyordu F4 / 80 (Klon: BM8) APC, anti-CD11c (Klon: N418) BV785. </li><li> Fixable Canlılık Leke ekleyin ve 5 dakika boyunca tezgah üstünde bırakın. Fixable Canlılık Stain fazlalığını ortadan kaldırmak için Sitometrisi Boyama Tampon çözeltisi ile hücreleri yıkayın. </li><li> Sabitleme / Permeabilization reaktif ekleyin ve 4 ºC de gece inkübe edin. permeabilizasyon çözeltisi içinde yıkanır. </li><li> hücre içi leke, 10 dakika süreyle, anti-CD16 / 32 FCR ile tekrar engeller. hücre içi lekeleri ekleyin ve 20-30 dakika inkübe edilir. pe yıkayın 2xrmeabilization çözeltisi. </li><li> FACS makine 3,9 ile Sitometrisi Boyama Tampon çözümü ve çalışma örneklerinde süspanse. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Biz burada güvenilir ve verimli bir şekilde böbrek bağışıklık hücreleri elde etmek için bir metodoloji sunduk. Yaygın olarak kullanılan kolajenaz sindirimi aşama (doku mekanik tahrip) başlıca modifikasyonu yaklaşık 30 dk tasarruf sağlar ve uygun bir bağışıklık hücreleri, çok sayıda yalıtım 4 Böbrek örnekleri için iki saatten az sürer. Ayrıca, bizim araştırma sorusu bağlı olarak, şimdi tek bir böbrek kullanmak bağışıklık hücre izolasyonu için (diğer böbrek Western lekeleri,...

Bağışıklık sistemi aktivasyonu çoklu böbrek hastalıkları ve patofizyolojik süreçlerde 6,10,11,13 oluşur. aktif bir araştırma için potansiyel alanlar, bağışıklık sistemi aktivasyonu için çeşitli tetikleyiciler kapsayan, çeşitli hücre türleri dahil, belirli bir hastalık ortamda sitokin / kemokin desen gibi, belirli bir ilaç tarafından bahsedilen işlemlerin tümünü modülasyonu iskemi-reperfüzyon olarak, Örnek vermek gerekirse, yaralanma (akut böbrek hasarı için bir model), (6 hafta sonra) 5 tamir veya fibrozis dönem boyunca sürekli bir kaç saat içinde hematopoietik hücreleri ya da CD45 + hücrelerinden oluşan bağışıklık hücreleri veya kemik iliği içinde bir artış vardır, 12. Bu bağışıklık hücreleri, tamir 5,12 işlemini düzenlemek için pro-enflamatuar ve anti-inflamatuar sitokinler ve kemokinler, hem salgılar. Şu anda, tek bir hücre süspansiyonu hücre popülasyonlarının etiket aynı anda birden fazla fluorophores kullanma yeteneği reklamla arttıdört beş lazerler ile makinelerin sitometri, modern akış havalandırma. Bu büyük ölçüde onların fonksiyonel durumu 3,7 dayalı hücre popülasyonlarının ayrımcılık yeteneği ekledi. F4 / 80, düşük CD11b yüksek Ly6b yüksek CD206, düşük, en az 3 fazla flüoroforlar canlı hücreler, CD45 + (lökositler) ve Ly6G- (nötrofiller) ve bulunmaktadır kapısı aynı numune hacmi içindeki gerekli olacaktır Örneğin, doğru bir makrofaj etiket Bu çok mümkün yeni akış sitometrelerinde 3 beraberdir. Ancak, sitokin salgılanması, hücre proliferasyonu, sitotoksisite, makrofaj aktivasyonu ve lenfositlerin ve monositlerin çeşitli alt kümeleri sayısı ölçümü için alt deneyler sadece kaliteli (canlı hücreleri, tekli), fakat hücrelerinin yeterli sayılarda gerekmektedir. Böbrek bağışıklık sistemi adaptif ve bileşenleri ve birden fazla hücre tipleri 1,7,13 hem oluşur. Örneğin farelerde, iki çocuk(1.4 x 10 6 hücre) ve bunların yaklaşık% 5-15 (1,400-4,200) izole edilmiş toplam böbrek bağışıklık hücrelerinin Neys birlikte 2-17% içerdiği rapor edilmiştir (28,000-266,000) CD45 + hücreleri, CD4 + hücreleri 1 olan , 5,12. Bu CD4 + hücrelerinin küçük bir yüzdesi (% 5-15, 70-630) FoxP3 + hücreleri (Şekil 1) 1 vardır. Nedeniyle (bu durumda CD45 + CD4 + FoxP3 + hücrelerinde) hücrelerin yüzdelerinde, bu adım adım azalmalar, ilgi konusu zaman hücre popülasyonuna 100 ° sadece hücreler tarafından temsil edilmektedir. CD45 + CD4 + FoxP3 + hücrelerinin az sayıda zorunludur toplam hücrelerin çok sayıda izole edilir ve hücreler, sitokin salgılama tahlillerinde alt-çalışmalar için iyi kalitede olmasını sağlar. Ayrıca, subpopülasyonunun ölçülebilir deneyleri gerçekleştirmek için yeterince yüksek sayıda temsil edilmemektedir çünkü 2-3 farelerin böbrek birleştirmek gerekebilir. Bu nedenle, böbrek mononükleer hücre popülasyonlarının güvenilir ve etkin izolasyon damızlık için arzu ediliry böbrek hastalıkları ile ilişkili immünolojik spektrum. Geleneksel olarak, böbrek mononükleer hücrelerin izolasyonu için, araştırmacılar, bu DNase 1 1,5,12 içeren kolajenaz 1A veya II gibi enzimatik sindirim çeşitli kullandık. İyi kollajenazlar uygun konsantrasyon ve inkübasyon 4,14,15 süresince titrasyonu zorunludur, lot numaraları ve imalat şirketi tarafından değişir enzimatik etkinliğe sahip olduğu bilinmektedir. Buna ek olarak, kollajenaz ile sindirim küçük parçalar halinde böbrek kıyma zaman ekler, reaksiyonun durdurulması için EDTA inkübasyon için ısıtılmış (37 ° C) banyo ve ek süre böbrek parçalarının inkübasyon gerektirmektedir. Buna ek olarak, daha az steril hücre kültürü ihtiyaç duyan bir alt işlemler için elde edilebilir. Daha da önemlisi, ilgili araştırmacı ve tüm değişkenlere bağlı olarak, bu laboratuarlar arasında veri ve yorumlanması değişkenlik yol açar. Son zamanlarda, ilemekanik doku bozucular / homojenizatör 16 geliştirilmesi, kolajenaz sindirimi aşama tamamen kaçınılması ve böbrekler 2 basit bir mekanik bozulma ile değiştirilir. Bu yazıda, gündelik bağışıklık hücre ekstraksiyon için böbrek bağışıklık hücrelerinin izolasyonu için basit ama etkili bir yöntem ortaya koymaktadır. Önemli olarak, bu teknik, örneğin karaciğer ve beyin 16 gibi diğer yumuşak ve lifsiz dokular adapte edilebilir.

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			<td>Stomacher 80 Biomaster lab system</td>
			<td>Seward</td>
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			<td>Stomacher 80 Classic bags</td>
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			<td>BA6040/STR</td>
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			<td>Sorvall Legend XFR Centrifuge</td>
			<td>Thermo Scientific</td>
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			<td>Electron Microscopy Sciences</td>
			<td>63514-11</td>
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			<td>Percoll&#160;</td>
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			<td>Dulbecco&#8217;s phosphate buffered saline 1X (DPBS)</td>
			<td>Gibco, Life Technologies</td>
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			<td>Polypropylene tubes, no cap</td>
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			<td>anti-CD45 (clone: 30-F11)&#160; BV421&#160;</td>
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			<td>Anti-Foxp3 (Clone: FJK-16s) APC</td>
			<td>EBioscience</td>
			<td>17-5773</td>
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			<td>Anti-CD127 (Clone: A7R34) PE/Cy7</td>
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			<td>Biolegend</td>
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			<td>Anti-Ly6G (Clone: 1A8) FITC</td>
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			<td>127605/6</td>
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			<td>Anti-CD11b (Clone: M1/70) PerCP-Cy5.5</td>
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			<td>101227/8</td>
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			<td>Anti-F4/80 (Clone: BM8) APC</td>
			<td>Biolegend</td>
			<td>123115/6</td>
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			<td>Anti-CD11c (Clone: N418) BV785</td>
			<td>Biolegend</td>
			<td>117335/6</td>
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			<td>Anti-CD301 (Clone: LOM-14) PE-Cy7</td>
			<td>Biolegend</td>
			<td>145705/6</td>
			<td></td>
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			<td>Anti-CD26 (Clone: H194-112) PE</td>
			<td>Biolegend</td>
			<td>137803/4</td>
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			<td>100 &#956;m filter&#160;</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
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			<td>Fisherbrand Tubes 50 ml</td>
			<td>Fisher</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
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			<td>Fisherbrand Tubes 15 ml</td>
			<td>Fisher</td>
			<td></td>
			<td>Or equivalent equipment&#160;</td>
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			<td>Sucrose</td>
			<td>Fisher chemical</td>
			<td>S5-3</td>
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			<td>Transfer pipette fine tip</td>
			<td>Samco Scientific</td>
			<td>232</td>
			<td>Or equivalent equipment&#160;</td>
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			<td>Flow Cytometery Staining Buffer Solution</td>
			<td>EBioscience</td>
			<td>00-4222-26</td>
			<td>Or equivalent equipment&#160;</td>
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		<tr>
			<td>1X RBC Lysis Buffer</td>
			<td>EBioscience</td>
			<td>00-4333-57</td>
			<td>Or equivalent equipment&#160;</td>
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reliable and high efficiency extraction of kidney immune cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

çalıştırılabilir panel sayısı güvenilir böbrekler üzerinden elde edilebilir bağışıklık hücrelerinin sayısına bağlıdır. Bu yazıda, 2 panel, T-lenfositlerinin için ve makrofajlar / dendritik hücreler için bir çalıştırma yeteneğini göstermek. T-lenfosit panelinde, ilk forward scatter (FSC) ve yan dağılım (SSC) şekline bakmak ve Şekil 1 (sol üst nokta arsa) &#39;de gösterildiği gibi ilgi nüfusu tasvir. Daha sonra, bir canlılık markö...

Watch this Scientific Journal Video about Reliable and High Efficiency Extraction of Kidney Immune Cells at JoVE.com

Reliable and High Efficiency Extraction of Kidney Immune Cells

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Böbrek Bağışıklık Hücreleri Güvenilir ve Yüksek Verimlilik Ekstraksiyon

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

reliable-and-high-efficiency-extraction-of-kidney-immune-cells

Research

JoVE Journal

Immunology and Infection

10.3K Görüntüleme.  Department of Medicine, University of Missouri-Columbia and Harry S Truman Memorial Veteran's Hospital. Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Video: Reliable and High Efficiency Extraction of Kidney Immune Cells

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 ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Bağışıklık Hücreleri murin aorta içinde Sitometrisi Analizi

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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 ...

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 ...

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 of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Böbrek gelen Çift Negatif αβ T Hücreleri İzolasyon

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran fare ve insan birincil NK hücreleri Lentiviral iletim verimliliğini artırır

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Isolation and Identification of Extravascular Immune Cells of the Heart

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 functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

Yalıtım ve kalp Extravascular bağışıklık hücreleri tanımlaması

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 ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

Plazma zarı verimliliği memeli hücrelerinde yeniden mühürlemeden yüksek üretilen iş ölçümü

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...