This work was supported by NIGMS, National Institutes of Health (GM094623). Significant portions of this work were performed in the Environmental Molecular Sciences Laboratory, a DOE/BER national scientific user facility located at the Pacific Northwest National Laboratory (PNNL). PNNL is operated for the DOE by Battelle under Contract DE-AC05-76RLO1830.

1. Advance hazırlayın <ol><li> 37 ° C sıcak bir steril fosfat tamponlu tuz (PBS). </li><li> % 10 fetal sığır serumu (FBS) ile desteklenmiş Eagle Medium (DMEM) ve Minimal Essential Medium (MEM) Sıcak Dulbecco modifikasyonu, 100 lU / ml penisilin ve 100 ug / ml streptomisin, 37 ° C arasındadır. Not: Bunlar sırasıyla RAW ve HeLa hücreleri için normal Büyüme Medya (NGM) temsil eder. </li></ol> Hücreler 2. Hazırlık <ol><li> NGM içinde% 70-90 confluency RAW 264.7 hücreleri büyümek. <ol><li> Nemlendirilmiş% 95 hava /% 5 CO2 atmosferinde, 37 ° C&#39;de hücreler muhafaza edin. </li></ol></li><li> NGM içinde% 70-90 confluency HeLa hücreleri büyür. <ol><li> Nemlendirilmiş% 95 hava /% 5 CO2 atmosferinde, 37 ° C&#39;de hücreler muhafaza edin. </li></ol></li><li> Toplanmadan önce steril PBS ile bir kez, hücre tekli tabakası yıkayın. </li><li> Steril konik bir tüp içinde önceden konfluent hücreler toplanır. <ol><li> Yavaşça RAW hücreleri kazıyınPBS içinde, bir lastik policeman ile tekrar pipetleme hücre toplamalardan dağıtmak için. </li><li> Görsel muayene kültür yüzeyinden ayrışma gösterene kadar% 0.25 tripsin solüsyonu ile HeLa hücreleri ayırmak. Örneğin, T-75 balonuna için 2-3 ml kullanılır. Tripsin çözüm tüm büyüme yüzeyini kaplayan sağlamak için buna göre ses seviyesini ayarlayın. <ol><li> NGM,% 10 FBS içeren ayrılma reaksiyonu söndürün. </li><li> Hücre toplamalardan dağıtmak için tekrar pipetleme ile, tripsin için NGM en az iki hacim kullanın. </li></ol></li></ol></li><li> Hafif 4 dakika boyunca 900 x g&#39;de santrifüj ile pelet hücreleri. </li><li> Steril PBS kullanarak tripsin / söndürme çözüm olarak aynı hacimde süspanse. </li><li> Hemasitometre veya parçacık sayacı kullanarak hücreleri saymak. </li><li> Hafif 4 dakika boyunca 900 x g&#39;de santrifüj ile pelet hücreleri. </li><li> 5,5 x 10 6 6 10 x 6.0 hücre / ml PBS yeterli hacimde süspanse. Not: Örneğin, bir T-75 şişe approxim verecektirulaştırılması 7.5 x 10 6 hücre 29. </li><li> Elektroporasyon kadar buz üzerinde hücre süspansiyonu tutun. </li></ol> Elektroporasyon 3. Hazırlık <ol><li> Buz Öncesi soğuk elektroporasyon küvetler (0.4 cm boşluk). </li><li> Elektroporasyon aparatı açın ve 0,3 kV gerilim ayarlayın. NOT: Kapasite ve direnç (üretici tarafından 10 iF ve 600 Ω ayarlı) bizim elektroporatörü ayarlanabilir ayarları değildi. </li><li> NGM ile geri kazanım plakaları, doldurun ve 37 ° C de, nemlendirilmiş bir% 95 hava /% 5 CO2 atmosferinde dengeye </li></ol> 4. Elektroporasyon <ol><li> Önceden soğutulmuş küvet içinde hücre süspansiyonu 400 ul koyun ve küvetin (ug / ml 50 ug) seçilen proteinin 20 ug ekleyin. </li><li> Flick küvet hafifçe ~ 10 kez hücrelere zarar vermeden karıştırın. Not: küvet de iyice karıştırın birkaç kez ters olabilir, ancak yukarı pipetlemeyin yok ve aşağı ya da vorteks hücreleri zarar vermemek için. </li><li&gt; Kağıt havlu veya diğer silecek ile küvetin kuru dış Elektroporatör elektrik arkı önlemek için. </li><li> 1.5-1.7 msn 0.3 kV electroporate örnek. Not: Bu, bu çalışma için tipik oldu. </li><li> Hemen elektroporasyon fiske küveti sonra yavaşça ~ 10 kat iyice karıştırın. </li></ol> Hücreler 5. Kaplama <ol><li> Hazır olana kadar buz üzerinde Electroporated hücreleri ile mağaza küvet kurtarma plakalarda yer. </li><li> En mansap analizler için, yabancı efektör protein kaldırmak için önceden ısıtılmış NGM ile hücreler 1x yıkayın. <ol><li> Analiz için hücreleri kaldırmak ve NGM 3-5 ml askıya. </li><li> 4 dakika boyunca 900 x g&#39;de santrifüj ile pelet hücreleri. </li><li> Istenen plaka boyutuna (örneğin 35 mm tabak için 2 mi) için NGM yeterli hacmi içinde süspanse. </li></ol></li><li> Mansap analizi için hücrelerin uygun miktarda çıkarın. <ol><li> Örnek 1: mikroskopi analizi için cam alt yemekleri içine plaka. </li><li> Örnek 2: levha intBöyle afinite saflaştırma gibi protein analizi için o hücre kültürü plastik. </li></ol></li><li> Hücrelerin en az 4 saat 37 ° C&#39;de nemlendirilmiş% 95 hava /% 5 CO2 atmosferde dengelenmiş plakalarda kurtarmak için izin verin. </li></ol> 6. Mikroskopi Analizi 6.1) Sabitleme / İmmünofloresan Boyama <ol><li> Yıkama hücreleri 4 saat iyileşme döneminden sonra steril PBS ile 1x. </li><li> Oda sıcaklığında 2 dakika boyunca% 100 metanol hücreleri saptamak. Tamamen hücreleri (örneğin 2 ml 35 mm çanak için) karşılamak için yeterli metanol kullanın. </li><li> Steril PBS ile yıkayın 3x. </li><li> 15 dakika boyunca PBS içinde% 0.4 Triton X-100 ile hücrelerin geçirgenliği. Örneğin, bir 35 mm çaplı levha 1 ml kullanılır. <ol><li> Epitop ile hedef proteinin konumuna göre, Triton X-100 geçirgenliği ve gücü uzunluğunu ayarlayın. Not: En iyi sonuçları ancak yukarıdaki koşullar çoğu c yeterli olmalıdır, ampirik her hedef için belirlenen gerekecektirytosolic hedefler. </li></ol></li><li> Oda sıcaklığında 1 saat için PBS içinde% 5 sığır serum Albumin (BSA) ile bloke edin. Örneğin, bir 35 mm çaplı levha 1 ml kullanılır. </li><li> PBS ile yıkayın 3x. </li><li> 4 ° C&#39;de bir gece boyunca antikor bağlayıcı çözelti içinde birincil antikor (PBS içinde% 0.1 Triton X-100 ve% 1 BSA) inkübe &#160; Hafifçe çalkalanarak ° C. Örneğin, bir 35 mm çaplı levha için 0,5 ml kullanılır. <ol><li> Antikor seyreltme için üreticinin tavsiyelerini uygulayın. Not: 200 seyreltme: PKN1 antikoru 1 kullanıldı olurken, 1000 seyreltme Örneğin, streptavidin bağlayıcı peptit etiketi (SBP-etiket) antikoru 1 kullanılmıştır. </li></ol></li><li> PBS ile yıkayın 4x. </li><li> Işıksız bir ortamda oda sıcaklığında 1 saat süre ile antikor bağlama çözeltisi içinde uygun floresan konjuge sekonder antikor ile inkübe edin. <ol><li> Örneğin, oda sıcaklığında 1 saat süre ile Alexa 488 veya Alexa 647 kullanır. <ol><li> Karıncanın üreticinin önerilerini izleyinibody seyreltme. (Örneğin, yaklaşık 1: 1000, bu çalışma için). </li></ol></li><li> Örneğin, gerekli olan diğer lekeler ekleme, 5 uM Buğday agglutinnin (WGA) ile oda sıcaklığında 1 saat süre ile, üreticinin tavsiyesine göre Alexa 647 veya DAPI birleştirilir. </li></ol></li><li> Görüntü hazır olana kadar ışıktan korundu PBS ile 4 ° C &#39;de deposu ile yıkayın 5x. </li></ol> 6.2) Konfokal Mikroskopi ve Görüntü Analizi <ol><li> 63x immersiyon yağı hedefi kullanarak bir ters konfokal mikroskop görüntü örnekleri. <ol><li> 492-542 nm arasındaki bant genişliği emisyonu ile argon lazer 488nm hattını kullanarak görüntü yeşil kanal. </li><li> 640-718 nm arasındaki bant genişliği emisyonu ile 633 nm diyot lazer kullanarak görüntü kırmızı kanalı. </li><li> 407-453 nm arasındaki bant genişliği emisyonu ile 405 nm diyot lazer kullanarak görüntü mavi kanal. </li><li> Z-yığınları hücresel hacmi bütününü kapsayan çoklu kanal emin olun. </li></ol></li><li> Uygun görüntü işleme yazılımı ile Süreç görüntüleri. </li></ol> 7. Affinity Arıtma <ol><li> 4 saat iyileşme süresinden sonra 4 ° C&#39;de iki kez PBS ile elektroporasyona hücreleri yıkayın. </li><li> Ile lizleyin ~ liziz tamponu buz üzerinde (PBS içinde proteaz inhibitör kokteyli ve fosfataz inhibitörü ile% 1 Triton X-100) ve 1.0 mi. Üretici talimatlarına göre inhibitörleri kullanın. Not: Örneğin, her ikisi de bu çalışmada kullanılan fosfataz ve proteaz inhibitörleri 100x de temin edilmiştir, ancak diğer formülasyonlar eşit derecede iyi çalışmasıdır. </li><li> Derhal lastik polis hücreleri kazıyın ve konik tüpler içine toplamak. </li><li> Güçlü döndürülerek ve sonikasyon ile lizleyin. Not: Diğer lizis yöntemleri eşit olarak iyi çalışabilir, ancak verimlilik ampirik tahlil gereksinimleri uygunluğu tespit edilecek gerekecektir. <ol><li> Aralıklı vorteks 3 x 30 sn sonikasyon. </li></ol></li><li> 4 ° C&#39;de 10 dakika boyunca 10,000 x g&#39;de santrifüjleyin toplamakHücresel enkaz ve çözünmeyen agrega; süpernatant kaydedin. </li><li> Sonu aşırı uç rotasyon ile bir gece boyunca 4 ° C &#39;de Streptavidin agaroz reçinesi süspansiyonuna 50 ul ile elektropore edildi hücre lizatının eşit hacimleri (~ 1.0 mi) birleştirin. Not: Bu reçine olan streptavidin bağlama peptidi afinite etiketi ile elektropore protein (ve ilişkili kompleksler) yakalar. </li><li> 2 dakika için 2.500 xg&#39;de Santrifüj ve süpernatant atın. </li><li> 40 yatak hacmi (~ 1 mi) iki kez PBS ile yıkayın. </li><li> 30 ul 4x LDS yükleme tamponu (141 mM Tris baz,% 2 LDS,% 10 gliserol, 0.51 mM EDTA, 0.22 mM SERVA Mavi G kırmızı 0.175 mM fenol, pH 8.5), 20 ul diH 2 O ve 1 ul ekle bir hacim sağlayan, 0.5 M tris (2-karboksietil) fosfin (TCEP); boncuk içinde kalması beklenir. </li><li> 10 dakika boyunca 95 ° C de ısı ve buz üzerinde soğutulur. </li><li> Sıkma 5 dakika boyunca 4 ° C &#39;de&gt; 10,000 xg. </li><li> Süpernatant toplayın. </li><li> Uygun antikorlar Not kullanarak batı leke gerçekleştirin: OYeniden anti-PKN1 birincil antikor kullanılır. </li></ol>

<ol>
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D., 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=Identification+of+GtgE,+a+novel+virulence+factor+encoded+on+the+Gifsy-2+bacteriophage+of+Salmonella+enterica+serovar+Typhimurium.">Identification of GtgE, a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium.</a> Journal of Bacteriology. 184, 5234-5239 (2002).</li><li>Niemann, G. 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=Discovery+of+novel+secreted+virulence+factors+from+Salmonella+enterica+serovar+Typhimurium+by+proteomic+analysis+of+culture+supernatants.">Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of culture supernatants.</a> Infection and Immunity. 79, 33-43 (2011).</li><li>Spano, S., Liu, X., Galan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+targeting+of+Rab29+by+an+effector+protein+distinguishes+the+intracellular+compartments+of+human-adapted+and+broad-host+Salmonella.">Proteolytic targeting of Rab29 by an effector protein distinguishes the intracellular compartments of human-adapted and broad-host Salmonella.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 18418-18423 (2011).</li><li>Haraga, A., Miller, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Salmonella+type+III+secretion+effector+interacts+with+the+mammalian+serine/threonine+protein+kinase+PKN1.">A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.</a> Cellular Microbiology. 8, 837-846 (2006).</li><li>Zinchuk, V., Grossenbacher-Zinchuk, O., 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=Quantitative+colocalization+analysis+of+confocal+fluorescence+microscopy+images.">Quantitative colocalization analysis of confocal fluorescence microscopy images.</a> Current Protocols in Cell Biology / Editorial Board, Juan S. Bonifacino ... [et al]. 4 (Unit 4.19), (2011).</li><li>Kimple, M. E., Sondek, 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=Overview+of+affinity+tags+for+protein+purification.">Overview of affinity tags for protein purification.</a> Current Protocols in Protein Science / Editorial Board, John E. Coligan ... [et al]. 9 (Unit 9.9), (2004).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Patojen bakterilerin salgılanan efektörleri konak hücre ortamında çalışabilmesi için geliştiğini ve böylece konak içinde in situ bunları incelemek yararlı olacaktır. Konakçı hücrelere özel ilgi efektörlerinin giriş ilgili patojen-konakçı etkileşimleri bakteriyel proteinlerden müdahalesi olmaksızın tek başına ele sağlar. amaç, böylece transfeksiyon ya da transdüksiyon ile bağlantılı bazı zorluklar kaçınarak ökaryotik konakçı hücrelere bakteriyel efektör proteinler kat?...

Bir çok Gram-negatif bakteriler, doğrudan konakçı hücreler 1-5 içerisine (efektör olarak anılacaktır) hastalık oluşturma ilişkili proteinler enjekte özel salgılama sistemleri kullanılmaktadır. Ev sahibi dokunulmazlık baskılanması, hücre iskeleti değişiklikler, hücre içi ticareti ve sinyalizasyon, transkripsiyon değişikliklerin modifikasyonu ve ev sahibi proteasome değişiklikler 6-9: Bu efektörleri dahil olmak üzere biyolojik fonksiyonları geniş bir yelpazesi var. Bazı efektörlerinin işlevleri ancak ev sahibi hedefleri ve diğerleri biyokimyasal eylem (ler) belirlenecek kalır bilinmektedir. Vahşi tip ve yeniden birleştirici bakteriyel enfeksiyonların karşılaştırılması hücre içi efektör hastalık oluşturma mekanizmaları incelemek için geçerli bir yaklaşım olsa da, konakçı hücre içerisine bağımsız bir efektör tanıtmak için sıklıkla yararlıdır. Böylece, konakçı hücreler bağlamında bakteriyel efektör proteinler katma ve karakterize edilmesi için basit bir yöntem arzu edilmektedir. Deneysel analiz wi BasitleştirmeTek bir efektör kritik inci diğer efektörler karşı ya da gereksiz işlevlere sahip olabilir. Bu basitleştirme gerçekleştirmek için, araştırmacılar, daha önce kazıma 12,13 yükleme viral transdüksiyon 10, mikroenjeksiyon 11 de dahil olmak üzere birçok farklı yöntemler ile hücrelere makromolekülleri getirmiştir, kimyasal olarak teşvik mikroenjeksiyon 14, özel bir protein &quot;transfeksiyon&quot;, reaktif 15, kalsiyum fosfat çöktürme ile hücre füzyonu 16 ve elektroporasyon 17-20. kişiye moleküller, hücre içi hedefleri 21,22 protein, hücre-geçirgen olmayan boyalar ve antikorlar, DNA, RNA ve RNAi türler dahil olmak üzere nükleik asitler arasında değişir. Bazı yöntemler dahil edilebilir makromolekül Çeşidi dahil kısıtlamaları vardır ve özellikle alt-analizler, yüksek hücresel toksisite, etki zarar mekanizmaları, düşük etkililik ve giriş verimliliği ile sınırlı olabilir. Transfeksiyon, bir ofteaynı zamanda makrofajlar ve birincil hücre gibi uygun bir ev sahibi hücre tipi, transfeksiyon karşı özellikle dirençlidir sınırlama uğrar, memeli hücrelerinde bakteri genleri ifade etmek için bir yöntem, n-kullanılmıştır. Bunun ötesinde, yabancı DNA&#39;nın sokulması üzerine üretilen bakteriyel protein seviyelerini kontrol etmek zordur. Çok iş bilinen bir laboratuvar tekniği olarak bakteriyel ve memeli yaratıkların hücrelerine nükleik asit elektroporasyon kurdu; Bununla birlikte, fizyolojik koşullar altında, hücre içine proteinlerin verilmesi için en iyi yöntem devam etmekte olan bir araştırma vardır. Protein transfeksiyon üzerindeki raporlar umut vericidir, ama pahalı reaktifler ve optimizasyon gerektirir. minimum maliyet ile hücre hedefleri çeşitli içine potansiyel toksik bakteri efektörler tanıtmak arzusu in vivo bu proteinleri eğitim için bir yöntem olarak elektroporasyon dikkate götürdü. Protein elektroporasyon 23-25 ​​bir araya geldi iseAyrıca elektro-transfeksiyon veya elektro-enjeksiyon 26 olarak bilinen electropermeabilization üzerinden canlı hücreler içine proteinleri tanıtmak hod. Bu teknik, hücre zarlarında gözenekleri oluşturmak için yüksek yoğunluklu elektrik darbeleri kullanır. Bu geri dönüşümlü gözenekler normal hücre içi alandan dışlanan makromoleküllerin hücreye girmesine izin. Dış elektrik alanının çıkarılması üzerine, zar, hücre gözenekler 27,28 geçirilir molekülleri korumak için izin kapatın olabilir. Standart bir Electroporator sürekli fare makrofaj-benzeri hücreleri ve insan epitel hücreleri hem de içine bakteri efektör tanıtmak için bu çalışmada kullanılmıştır. yöntem hücresel canlılığı hiçbir kayda değer azalma ile, hızlı, verimli ve ucuz. kişiye proteinler immünofloresan mikroskopi ile görüntülenmiştir veya fonksiyonel deneyleri için de kullanılabilir. Bu aynı zamanda, bir toksik-olmayan, standart olarak yeşil floresan proteini (GFP) ile gösterilmiştirİki Salmonella efektör proteinler, SspH1 ve GtgE. Bu ökaryotik konakçı hücrelerde bu bakteriyel hastalık oluşturma proteinleri ve işlevlerin çalışması için repertuarında ek bir araç olarak, protein elektroporasyon önerilmektedir.

<table border="0" cellpadding="0" cellspacing="0" width="1132">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">Material or Equipment</td>
			<td style="width:177px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:315px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.25% Trypsin-EDTA Solution</td>
			<td>Cellgro</td>
			<td>25-050-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4cm Gap-disposable electroporation cuvettes</td>
			<td>Bio-Rad</td>
			<td>165-2088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100% Methanol</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Flammable, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A4919</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Counting Apparatus - Hemocytometer or Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>Model Z1</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;">Cell Culture Incubator</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Humidified 95% air/5% CO2 atmosphere at 37 &#176;C&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Cell Culture Plastic</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Cell culture flasks/plates, pipets, tubes, rubber policeman</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Modification of Eagle&#8217;s Medium (DMEM)</td>
			<td>Cellgro</td>
			<td>10-013</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Electroporator</td>
			<td>Bio-Rad</td>
			<td>E. coli Pulser</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-016-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent confocal microscope&#160;</td>
			<td>Ziess</td>
			<td>Model&#160; LSM 710&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Bottom Dishes for Microscopy</td>
			<td>Wilco Wells</td>
			<td>HBSt-3522</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HALT Protease Inhibitor Cocktail</td>
			<td>Pierce</td>
			<td align="right">78430</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HeLa Cell Line</td>
			<td>ATCC</td>
			<td>ATCC CCL-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LDS 4X Loading Buffer</td>
			<td>Invitrogen</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimal Essential Medium (MEM)&#160;</td>
			<td>Cellgro</td>
			<td>10-010</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Other fluorescent stains (WGA, DAPI) in conjunction with anti-fade reagent</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RAW 264.7 Cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primary Antibody Against Target of Interest</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Secondary Antibody Conjugated to Fluorophore</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Chill to 4 &#176;C&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Phosphate Buffered Saline</td>
			<td>Any</td>
			<td>N/A</td>
			<td>Warm to 37 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Agarose Resin Suspension&#160;</td>
			<td>Pierce</td>
			<td align="right">20353</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge Capable of Accepting Conical Tubes (swinging bucket preferred)</td>
			<td>Any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TCEP</td>
			<td>Sigma-Aldrich</td>
			<td align="right">646547</td>
			<td>Corrosive, Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8585</td>
			<td>Irritant, Toxic</td>
		</tr>
	</tbody>
</table>

electroporation of functional bacterial effectors into mammalian cells

canlı hücrelerin bağlamında protein etkileşimleri çalışma lokalizasyonu, dinamikleri ve etkileşim ortakları hakkında kritik bilgiler üretebilir. Bu bilgiler, evsahibi-patojen etkileşimlerinin bağlamında özellikle değerlidir. Birçok patojen proteinleri, hücre içi bir ortam içinde konak bağışıklık sistemi ve hayatta kalma kaçırma sağlayan, bu gibi bir şekilde çeşitli konak hücreleri içinde çalışır. Bu patojenin protein konukçu hücre etkileşimini incelemek için çeşitli yaklaşımlar sık dahil olmak üzere kullanılır: in vivo enfeksiyon transfeksiyon ya da transdüksiyon ile etiketli veya mutant protein ya da patojen genlerin katılması eksprese eden bir suşu ile. Bu yaklaşımların her biri avantajları ve dezavantajları vardır. Bu direkt olarak hücrelere ekzojen proteinleri katılması için bir araç çalıştı. Elektroporasyon yaygın hücrelere nükleik asitlerin dahil edilmesi için kullanılabilir, ancak biyo-fiziksel temeli tam olarak aynı olmasına rağmen daha nadir proteinlere uygulanmıştır.Standart bir Electroporator memeli hücrelerine afinite etiketli bakteriyel efektör tatbik etmek için kullanılmıştır. İnsan epiteliyal ve fare makrofaj hücreleri, geleneksel metodlarla kültürlenmek sureti ile ayrılır ve ilgi dahilindeki bir eksojen bakteriyel patojenin proteini (örneğin, Salmonella Typhimurium GtgE) ile 0.4 cm boşluk elektroporasyon küvetler içine yerleştirilmiştir. Elektroporasyon (0.3 kV) ve kısa (4 saat) geri kazanım süresinden sonra, hücre içi protein, flüoresan olarak afinite etiketi ile protein etiketleme ve konfokal mikroskopi ile mekansal ve zamansal dağılımının incelenmesi ile doğrulanmıştır. Elektroporasyona protein aynı zamanda hücre içinde işlevsel ve doğru hücre içi ticareti ve protein-protein etkileşimi yeteneğine sahip olduğu gösterilmiştir. Ekzojen proteinler hücrelerin yüzeyi üzerinde birikme eğiliminde iken, elektroporasyona örnekler sadece kuluçkalama için hücre içi efektör konsantrasyonda büyük artışlar vardı. protokol ap yapılacak basit ve yeterince hızlıhücre içi hedefleme ve hastalık oluşturma proteinlerin işlevine de dahil olmak üzere, konakçı hücrelerde patojen proteinlerinin yüksek verimli bir karakterizasyon için izin arallel moda.

Kavramının bir ilk kanıtı olarak, saflaştırılmış yeşil flüoresan proteini başarıyla elektroporasyon ile memeli hücrelerine dahil edilmiştir. GFP, yaklaşık 27 kD molekül ağırlığına sahip protein özel genellikle önemli hücresel toksisite olmaksızın moleküler biyoloji aracı olarak (normal olarak plasmid DNA ifade edilen), memeli hücreleri içine dahil edilir. HeLa hücreleri (Şekil 1A) kuluçkalanmıştır veya floresan GFP sinyali kontrol etmek için imüno-konfokal mikrosk...

Watch this Scientific Journal Video about Electroporation of Functional Bacterial Effectors into Mammalian Cells at JoVE.com

Electroporation of Functional Bacterial Effectors into Mammalian Cells

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Memeli Hücreleri içinde fonksiyonel Bakteriyel Efektör elektroporasyonu

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

electroporation-of-functional-bacterial-effectors-into-mammalian-cells

Research

JoVE Journal

Immunology and Infection

10.0K Görüntüleme.  Pacific Northwest National Laboratory. Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells. 

Video: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Bakteriyel Patojen İnsan Hücreleri Invasion

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

İmmünoloji ve Enfeksiyon

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

Bakteriyel Yapışma Memeli Epitel Hücreleri üzerine In Vitro Testi

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Miyeloid hücrelerin Dynamics Gerçek zamanlı Görüntüleme<em&gt; Süm<sup&gt; Min / +</sup</em&gt; Bağırsak Tümörleri Disk Eşodaklı Mikroskopi Spinning tarafından

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periyodik Asit Schiff Boyama ile periferik kan mononükleer hücreleri glikojen algılama

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Üretim ve Çok fenotipik Yüksek içerik Eleme<em&gt; Coxiella burnetii</em&gt; Transposon Mutantlar

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Bakteriyel Yapay Kromozom: RNA Virüsler Pozitif iplikli Araştırmaları Fonksiyonel Genomik Aracı

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Bir Geçirgen Membran Ekle tabanlı Enfeksiyon Sisteminin Uygulanması memeli konakçı hücreleri üzerinde salgılanan Bakteriyel Toksinler Etkilerinin Eğitim için

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Bir Maya İki hibrid Ekran kullanarak Olmayan ekilebilir Bitki Patojen bir Bakteriyel Efektör Fonksiyonu aydınlatmak

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

Memeli Hücrelerinin Bakteri Mücadelesinden Sonra Stres Granül Oluşumunun İmmünofloresan Analizi

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...