Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

Makrofaj Hücre Hatları 1. Bakım <ol><li> RAW264.7 Büyüme ve DMEM içinde J774A.1 hücre hatları,% 5 CO2 varlığında 37 ° C&#39;de% 10 fetal sığır serumu (FBS) ile takviye edilmiştir. , Kısırlık korumaya yardımcı (bu kesinlikle gerekli olmasa da) medyaya% 1 penisilin / streptomisin (pen / strep) ekleyin. </li><li> Kritik adım: makrofajlar verimli nakil ve siRNA kaynaklı gen demonte için sağlıklı bir durumda büyüyen emin olun. Bu makrofaj soylarında altında en iyi transfeksiyon verimi sergiler ve bu aşamada sağlam post-transfeksiyon yaşamının devam taze, çözüldükten sonra geçitler 3 ve 10 arasında hücreleri kullanmak; de büyük ölçüde azalır demonte transfeksiyon verimi ve gen gibi, daha yüksek bir geçiş numarası (&gt; 20) kullanmayın. </li><li> Hücreler, en fazla% 80 kaynaşmaya eriştikten kadar optimize siRNA Transfeksiyon için, levha 20% konfluansta pleytlendi hücreleri ve bir ya da iki gün büyür. Büyümüş hücreleri effic transfekte olmaziently. </li></ol> 2. Transfeksiyon için 96 oyuklu servisi sistem programlama <ol><li> Mekik bağlı bilgisayarda 96-iyi servis yazılımını başlatın. Sol üst dosya menüden yeni bir parametre dosyayı seçin. </li><li> Görüntülenen 96 oyuklu plaka şekil üzerine transfekte edilecek kuyu Vurgu. Yeşil bir kutu seçilir hangi kuyular gösterecektir. </li><li> Diagramed 96-plaka altında bulunan (J774A.1 için RAW264.7 veya DS-130 DS-136) kullanılan ve uygulamak seçeneğini makrofaj hücre hattı, karşılık programı seçin. Bir programı seçtikten sonra, şematik plaka üzerinde kuyu koyu mavi renkli olacaktır unutmayın. Boş çevreler bir program atanmış değil ve şok olmayacaktır kuyu gösterir. </li></ol> 3. Transfeksiyon için, reaktiflerin hazırlanması <ol><li> SF hücre hattı karıştırılarak çalışma Nucleofector ™ çözeltisi hazırlayın 96 oyuklu Nucleofector ™ çözümükapalı ek tüpün tüm içeriği ile. Karışık reaktifler, kullanılmadan önce oda sıcaklığında dengelenmeye bırakın. Ileride kullanmak üzere 3 aya kadar 4 ° C&#39;de aşırı komple Nucleofector ™ çözümü saklayın. </li><li> Ön sıcak transfeksiyonu için makrofajlan hazırlamadan önce 37 ° C&#39;de DMEM, RPMI-1640,% 0.25 tripsin / EDTA, ve fosfat tamponlu tuz (PBS). </li><li> Buz üzerinde eritilmesi ile transfeksiyonu için siRNA hazırlayın. SiRNA donma çözülür sayısını en aza indirmek için, alikotlarda kullanıldıkları ilk kez yeni siRNA&#39;ları dondurmak. </li><li> Başlangıç ​​deneylerinde, (20 uM stoktan 1:10 seyreltilmiş) 2 uM nispeten yüksek bir dozda siRNA kullanın. Tipik olarak daha yüksek dozlar diğer hücre tipleri ile karşılaştırıldığında, makrofajlarda etkili gen demonte için gerekli olmasına rağmen sonra, daha düşük dozlar aşağı doğru demonte sağlam genini endüklemese siRNA&#39;lar titre edilir. NOT: siRNA&#39;lar birçok kaynaktan mevcuttur; Oldukça sağlam gen demonte elde edilebilirHer genini hedefleyen dört siRNA&#39;ların havuzları siGenome SMARTPOOLs kullanarak. Bir negatif kontrol olarak, çeşitli non-hedefleme siRNA havuzları veya siRNA&#39;lar herhangi birini kullanabilirsiniz. </li><li> , Transfeksiyon verimini izlemek deney siRNA&#39;lar paralel pmaxGFP kontrol (Amaxa kitlerinde) plazmid veya floresan ile etiketlenmiş siRNA&#39;lar ya da transfekte etmek. GFP plazmidi% 30-50 transfeksiyonu (mikroskopi ile analiz edilir veya transfeksiyondan bir gün sonra akış sitometrisi) ve (transfeksiyon ve geri kazanımdan sonra sitometrisi hemen akış ile tahlil), flüoresan-işaretli siRNA&#39;nın&gt;% 99 transfeksiyon bekliyoruz. </li></ol> 4. Transfeksiyonu için makrofajlan hazırlanması <ol><li> Tripsinizasyon ile hücre kültür tabaklarından yapışkan makrofajlar ayırın. Ortamı aspire ve 10 ml PBS ile iki kez hücreleri yıkayın. PBS uzaklaştırıldıktan sonra, hücreler ile inkübe hücreleri ayırmak alıncaya kadar 1-2 dakika boyunca% 0.25 tripsin / EDTA (1 mi / 10 cm plaka), önceden ısıtılmış. Yavaşça plaka dokunun ve Solu hareketinkübasyon sırasında siyon tripsinizasyon maksimize etmektir. </li><li> İnkübasyondan sonra, steril bir tüpe pipetleme hücreleri, 9 mi DMEM FBS ortamı ilave yukarı ve aşağı, yavaşça karıştırın ve toplayın. </li><li> Hemasitometre kullanarak izole makrofajlar sayısını. 10 cm plaka başına kabaca 10 milyon makrofajlar bekliyoruz. Gerekli makrofaj sayısı hesaplanır; iyi 96 kuyu mekik her 200.000 hücreleri gerektirir. Pipet kayıplar için izin vermek hücrelerin fazladan birkaç kuyu değerinde eklemeyi unutmayın. </li><li> Bir santrifüj tüpüne hücre hesaplanan sayısı Pipet. Oda sıcaklığında, 150 x g&#39;de 10 dakika boyunca hücreler santrifüjleyin. Bu makrofaj sağlık ve transfeksiyon verimliliği azaltacak gibi çok hızlı santrifüj etmeyin. </li></ol> SiRNA ile Makrofagların 5. Nucleofection <ol><li> (Tepkin madde kaybını minimize ederken karışmasını kolaylaştırır), steril bir 96-gözenekli yuvarlak tabanlı bir plaka kullanarak, her bir oyuğa olduğu transf her siRNA 2 ul ekleandırılmaktadır. Not: siRNA&#39;lar sayısına bağlı olarak, bu makrofaj santrifüj aşaması esnasında gerçekleştirilebilmektedir. </li><li> Santrifüj hücrelerden ortam aspire. Yavaşça peletlendi 200.000 hücre başına 20 ul kullanarak, (ek solüsyon içeren) Nucleofector ™ çözümü SF hücreleri tekrar süspansiyon. Yeniden süspansiyon haline sonra, en iyi transfeksiyon için hemen hücreleri kullanır. </li><li> Steril bir çukur içine süspanse hücreler aktarın; siRNA&#39;lar ihtiva eden 96 oyuklu bir plakanın her bir oyuğuna transfer yeniden süspansiyon haline getirilmiş hücre karışımı 20 ul, bir çok kanallı pipet kullanarak. Hafifçe yukarı ve aşağı pipetleme karıştırın. </li><li> Transfer 96 oyuklu Nucleocuvette ™ plaka içine hücre / siRNA karışımı 20 ul Kritik adım.: Bu nucleofection sırasında hataları indükleyebileceği transfer adımı sırasında kabarcıkları sakınmayı. </li><li> Transfeksiyon plakasında Deney örneklerinin her yerleştirildikten sonra, Resim kapak ve ver 96 oyuklu plaka kapağıy yavaşça örneklerinde herhangi bir kabarcıklarını çıkarmak için sert bir yüzeye dokunun. </li><li> Program ekranın sağ alt &#39;upload ve start&#39; 96 kuyu Nucleofector mekik içine kapaklı 96-plaka yerleştirin ve seçin. Nucleofection önce, sonuçları kaydetmek için program istemini izleyin. Not: Nucleofection sürecinde, başarıyla tranfected kuyular bir artı işareti ile yeşil bir daire ile ekranda 96-kuyu şematik tasvir edilecektir. Eksi işaretleri ile kırmızı bir daire kabarcıkların veya mis-pipetlenmesinden reaktiflerin doğru hacmi için, büyük olasılıkla bir hata gösterir. </li></ol> 6. Kurtarma ve Makrofagların Kaplama <ol><li> Hemen nucleofection sonra, çok kanallı bir pipet kullanılarak, her bir oyuğa RMP1-1640 ortamı önceden ısıtılmış (37 ° C) 80 ul ekle. Ortam yavaş yavaş bu aşamada çok hassas olan transfekte edilmiş hücreler ile karışmasına imkân verilmesi, her bir oyuğa tarafına ortam ekleyin. Bu irade gibi, çok hızlı ortam eklemeyinbüyük ölçüde canlılığını azaltır. </li><li> 2 dakika boyunca% 5 CO2 içeren bir 37 ° C inkübatör içine transfekte edilmiş numuneleri ile RPMI-1640 ile 96 oyuklu plakalar yerleştirin; Bu hücrelerin manipülasyonu daha ileri önce kurtarmak için sağlamak açısından önemlidir. </li><li> Inkübatör 96 oyuklu plaka çıkarın ve dikkatli bir şekilde bir çok kanallı pipet kullanılarak 96 oyuklu bir doku kültür plakasının her bir kuyudan karışımı aktarın. </li><li> Bir çok kanallı pipet kullanarak, her bir oyuğa önceden ısıtılmış (37 ºC), DEM + FBS orta 100 ul ekleyin. </li><li> 24-36 saat boyunca% 5 CO2 içeren bir 37 ° C kuluçka makinesi içinde örnekleri ile 96 oyuklu doku kültürü plakası (bu zaman aralığı genel olarak iyi çalışmasına rağmen, tek tek siRNA&#39;lar için tam zamanlama optimize) inkübe edin. </li></ol> LPS ile Makrofagların 7. uyarılması <ol><li> 24-36 saat sonra-Nucleofection inkübe edildikten sonra, taze ortam içinde LPS ile (ya da diğer üzerine durmaktadır) maddesinin hazırlanması. Edilen kuyuların tümü için yeterli taze ortam hazırlayın (200 ul m,edia / oyuk, 96 oyuklu plaka artı pipetleme için fazladan). DMEM FBS içinde 20 ng / ml&#39;lik nihai bir konsantrasyona kadar LPS seyreltin. </li><li> 96 gözlü levhadan dikkatlice aspire ortam ve hücreler LPS içeren taze ortam ilave edin. </li><li> Stimülasyon sonrasında çeşitli zamanlarda LPS veya diğer üzerine durmaktadır yanıtı izlemek. 6 saat, IL-6 veya TNFa gibi sitokinler için sağlam bir enflamatuar sitokin tepkisi meydana getirmek için yeterlidir. </li></ol> 8. Makrofagların yılında Başlatılan Doğuştan Bağışıklık Tepkisi, Gen demonte ve canlılık İzleme <ol><li> LPS-kaynaklı sitokin üretimini izlemek için, 96-çukurlu bir depolama plakasına hücrelerden süpernatant pipetle. Sitokin üretimi daha sonra ELISA ile ölçülebilir. </li><li> Süpernatan kaldırıldıktan sonra, floresan diasetat kullanılarak hücre yaşamı sürdürme kabiliyetini izlemek (canlı hücrelerde hücresel esterazlarla ayrılır zaman, bu bileşik, flüoresan hale gelir). T kullanılarak hücre canlılığı kontrol eden genleri hedef siRNA&#39;ları tespitOnun yaklaşımı. Not: düzgün yapıldığı takdirde transfeksiyon sürecinin kendisi canlılığı üzerinde çok az etkiye sahip olması gerekmektedir. <ol><li> (: 100, PBS içinde nihai sulandırma 1), her bir sıra için floresan diasetat (aseton içinde 5 mg / ml stok) ilave et. </li><li> Beş dakika için bir oda sıcaklığında inkübe edin ve bir plaka okuyucusu (460 nm eksitasyon, 515 nm emisyon) kullanılarak hücrelerdeki floresan izler. </li><li> İsteğe bağlı: kuyularda floresan homojen olduğundan emin olmak üzere, bu gibi RIPA gibi tamponlar kullanılarak lize hücreleri; Bazı levha okuyucular de sadece küçük bir bölümünü izlemek için yapılandırılmış olan bu, kuyunun içinde üniform floresan sinyali sağlar; hücreler eşit kaplama değilse, floresandiasetat bir yanlış okuma verebilir. </li></ol></li><li> Hücre canlılığı izlenmesi için bir alternatif olarak, yapışan hücrelerden türetilen RNA kullanılarak qPCR demonte siRNA teşvik etmiş geni izler. <ol><li> Süpernatanı, hücrelerden çıkarılır sonra reklam (yukarıda 8.1 adım a)bunları eritmek doğrudan yapışık hücrelere (liziz ve RNA korunması için kullanılır) D RLT tamponu. </li><li> Örneğin, RNeasy Mini kit gibi bir RNA hazırlama kiti kullanılarak RNA izole edilir ve gen siRNA tedavi kontrolü için demonte göreceli izlemek için gen spesifik primerler ile qPCR kullanın. Normalleşmesi için Βactin için astar veya GAPDH kullanın. </li></ol></li><li> Başka bir alternatif ise, doğal immun cevabın başka yönlerini izlemek için de, sonra ya FITC-etiketli E. ekleyerek makrofagların fagositik yeteneği izlemek doğrudan bir kit kullanılarak yapışık hücreler üzerine E. coli parçacıklar. Sadece birkaç saat sürer üreticinin protokolünü takiben, bir plaka okuyucu veya mikroskopi kullanılarak fagositik alımını izlemek. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim. Bu makale Lonza&#39;nın, Inc. tarafından sponsor oldu

Çok sayıda çalışma, burada tek tek genler sıçangil makrofaj siRNA tarafından hedef alınmıştır yayınlanmıştır. Lipit aracılı transfeksiyon bireysel olarak makrofaj hücre hatlarına siRNA teslim etmek için kullanılmış olsa da, bu yöntemler, bir genden genine canlılığı, sınırlı gen demonte ve değişkenlik olası etkileri muzdarip. Orta veya yüksek verimli bir şekilde genlerin hedeflenmesi için kullanılabilecek daha sert tahlilleri geliştirmek için, gen demonte içinde daha fazla kıvam...

Bu protokol, biz siRNA&#39;ları kullanarak fare makrofaj hücre hatlarında genleri inhibe ve doğal immün yanıt üzerindeki bu tedavilerin etkilerinin izlenmesi için etkin yöntemler açıklanmaktadır. Bu prosedürler, orta ya da yüksek verimli bir şekilde RNAi ekranlar için izin veren, 96-kuyu formatında gerçekleştirildi. Enfeksiyona yanıt olarak, insanlar hemen doğuştan gelen bağışıklık tepkisini daha yavaş olan ancak daha özel bir uyum sağlayıcı bağışıklık tepkisi. Bu hızlı doğal immün yanıt makrofajlar 1 olmak üzere fagositik doğuştan gelen bağışıklık hücreleri toplanmasını ve aktivasyonunu içerir. Klasik olarak aktif makrofajlar, akut enflamatuar yanıtlarda rol oynayan ve antimikrobiyal proteinlerin ve bileşiklerin, sitokinler ve kemokinleri ve antijenlerini 2,3 üretirler. Alternatif aktive makrofajlar hoşgörüyü, doku onarımı bakımı, bağışıklık düzenlenmesinde rol oynar ve 4-8 yara iyileşmesi. Çünkü işlevleri onların geniş dizi, Makrofajlar ateroskleroz, arterit, kanser ve 9 da dahil olmak üzere pek çok hastalıkta önemli bir rol oynayabilir. Bu nedenle, makrofajların çalışma hastalık alanları arasında bir yelpazede bir süre için bir araştırma anahtar alanı olmuştur. Doğuştan gelen bağışıklık tepki olarak önemine rağmen, makrofajlar ile çalışmak için hücreler zor olabilir. Özellikle, toksisitesi, 10,11 makrofajların lipid reaktifler kullanılarak verimli transfeksiyona elde etmek zordur. Ayrıca, siRNA verimli makrofajlar teslim olduğunda bile, genellikle demonte RNAi neden geninin sağlamlığı oldukça ılımlı olabilir ve gen için gen arasında değişebilir. Bu teknik zorlukları aşmak için, iki fare makrofaj hücre hatları, RAW264.7 17 ve J774A.1 18 transfeksiyonuna ve demonte teknikleri 12-16 optimize ettik. Bu yaklaşım, transfeksiyon için Amaxa Nucleofector 96 oyuklu Servisi Sistemi kullanır; bu sistemin96-çukurlu formatta 19 hücreleri transfekte etmek için özel reaktifler ve elektroporasyon bir kombinasyonunu kullanır. Transfeksiyondan ardından, yöntem hücresi geri kazanımı ve canlılığı maksimize etmek ve daha sonra, siRNA ile uyarılan gen demonte üst düzeye çıkarmak için tarif etmektedir. Bu yaklaşımın faydasını göstermek için, lipopolisakkarit (LPS) ile bu hücreleri uyarır ve çeşitli pro-enflamatuar sitokinlerin üretim düzeyinde doğuştan gelen bağışıklık tepkisinin izlenmesi, aşağıdaki makrofaj hücre hatlarına siRNA taşınması için bir protokol açıklar. Biz üyeleri LPS ve diğer patojen ilişkili moleküler şekilleri (üzerine durmaktadır) duygusu, doğuştan gelen bağışıklık düzenleyen Toll-benzeri reseptör (TLR) ailesi, hangi hedef örnek veri sağlar.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

using rna-interference to investigate the innate immune response in mouse macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

Bu yaklaşımı kullanarak transfeksiyon etkinliğini göstermek için, bir akış sitometresi (Şekil 1) kullanılarak, FITC-işaretli siRNA&#39;nın alımını izlenir. Doğuştan gelen bağışıklık yanıtı izlemek için bir yaklaşım kullanışlılığını tasvir etmek için, biz, RAW264.7 makrofaj hücre hattı bilinen doğuştan gelen bağışıklık düzenleyici genler hedef siRNA&#39;lar transfekte LPS ile stimüle edilen hücreler, daha sonra pro-enflamatuar si...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Fare Makrofagların yılında Doğuştan Bağışıklık Tepkisi Soruşturma RNA girişimi kullanma

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

using-rna-interference-to-investigate-innate-immune-response-mouse

Research

JoVE Journal

Immunology and Infection

10.6K Görüntüleme.  National Jewish Health and University of Colorado School of Medicine. In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Video: Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

Keneler de RNA Girişim

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

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 ...

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 ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

İmmün Yanıt ve Uyku içinde kantitatif ölçümü<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Zebra balığı Doğuştan Bağışıklık Sağlık Bir Göstergesi Olarak Solunum Burst Tepki Kantitasyonu

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Sitokin Üretimi Viral Evasion Doğuştan Bağışıklık Sinyalizasyonu Diseksiyon

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Batı Nil Virüsü Mutant Soy Enfeksiyon MyD88-aracılı Hücresel Bağışıklık Tepki Vitro Analizi

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Bir Zebra balığı Larva Modeli Doğuştan Bağışıklık Tepki Non-invaziv Görüntüleme<em&gt; Streptococcus iniae</em&gt; Enfeksiyon

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Ökaryotik gen kökenli araştırmaya filogenetik analizi kullanılarak

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Fare Makrofagların yılında Doğuştan Bağışıklık Tepkisi Soruşturma RNA girişimi kullanma

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