Name: silac based proteomic characterization of exosomes from hiv-1 infected cells
Uploaded: 2017-03-03T12:30:00.000+00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells at JoVE.com

Bu çalışma BR ömrü / Tufts / Kahverengi SYAO, P30AI042853-13S1, NIH P20GM103421, P01AA019072, R01HD072693 ve K24HD080539 bir arra ek tarafından desteklenmiştir. Bu çalışma aynı zamanda ömrü Pilot Araştırma Fonu (# 701- 5857), Rhode Island Vakfı Tıbbi Araştırma Grant (# 20133969), ve ML NIH COBRE URI / RİH Pilot Araştırma Grant (P20GM104317) tarafından desteklenmiştir. Biz el yazması ve şekil hazırlanması ile ilgili yardım için, James Myall ve Vy Dang teşekkür ederiz.

1. Hücre Kültürü ve HIV-1 enfeksiyonu NOT: deneyler başlamadan önce, bir MTT testi 12 ile Tripan Mavisi boyaması 11 ile hücrelerin canlılığı ve çoğalmasını kontrol edilmesi tavsiye edilir. Aynı zamanda yeni hazırlanan SILAC ortamını kullanmak için kritik öneme sahiptir. Çeşitli hücre çizgileri de aktif olarak çoğalma aşamasında olan ve HIV-1 enfeksiyonu, veya tercih test koşulu duyarlı olduğu sürece, kullanılabilir. Bu protokol, örnek olarak H9 hücre hattı kullanın. <ol><li> Her bir hücre kültür şişesi içine Tohum 2 x 10 6 H9 hücreleri. % 10 diyalize edilmiş fetal büyükbaş hayvan serumu (FBS), 100 mg / L &#39;13C 6 L-lizin ve 100 mg / L 13 C 6 15 N4, L-arginin ihtiva eden RPMI 1640 ortamında etiketli 10 ml bir grup büyütün. FBS% 10 diyalizlenmiş, 100 mg / L L-Lizin ile 100 mg / L L-arginin ile, 10 ml işaretlenmemiş RPMI 1640 ortamı diğer grup büyütün. </li><li>Ağır amino asitler ile etiketlenmiş ortam içinde hücrelerin proteinler, hemen hemen tamamen etiketlenir ve bu noktada altı katlanmaya (&gt;% 99) hücreleri büyütün. taze medya ekleyin veya düzenli ortamı değiştirmek (her 1-3 gün hücre tipine bağlı olarak. H9 hücre için, orta her 3 günde değiştirme). Etiketleme sonunda, daha fazla hücre büyümeyi kültür hacmi (örneğin ~ 30 mi) artar. Not: Tripan Blue lekelemesi ve hücre sayımı ile deneyin başlangıcında ikiye katlanma süresinde tam hücre belirler. </li><li> HIV-1 NL4-3 48 saat boyunca standart bir HIV-1 enfeksiyonu protokolü 13 ile etiketlenmiş hücreleri enfekte etmektedir. 0.3 civarında bir enfeksiyon çokluğu (MOI) virüs uygun miktarlarda hücreleri inkübe edin. Bir HIV-1 p24 antijen ELISA kiti kullanılarak kültür süpernatanlarının p24 miktarının HIV-1 enfeksiyonu kontrol edin. Hücrelerin başarılı enfeksiyonu teyit etmek için bir immünofloresan tahlil 14 gerçekleştirin. Keep, HIV-1 enfeksiyonu olmayan etiketlenmemiş ortam içinde etiketlenmemiş hücrelerin büyüyen. </li><li> enfeksiyon sonunda (adım 2.1) de her iki grubun süpernatantlar hasat. </li></ol> 2. Exosome İzolasyon Not: ultrasantrifügasyon bir dizi aşama ile, kültür süpernatanlarından exosomal kısımlar 15 zenginleştirilmiştir. En az 100,000 x g hızda ulaşabilir Ultrasantrifüj rotoru ile 4 ° C &#39;de, aşağıdaki adımları gerçekleştirir. <ol><li> Hücreleri kaçınarak, 50 ml konik tüplerde kültürlerin süpernatantlar toplayın. kalan hücrelerin çıkarılması için, 300 x g&#39;de 10 dakika boyunca santrifüj. </li><li> Yeni 50 ml konik tüplerde süpernatantlar toplamak ve ölü hücreleri çıkarmak için 2,000 x g&#39;de 10 dakika boyunca santrifüj. Ultrasantrifügasyon dayanabildiğinden ticari rotor uyumlu tüplere süpernatantlar elde edilen transfer. </li><li> ultrasantrifüjdeki tüpleri dengelemek için emin olun. çıkarmak için 10,000 x g&#39;de 30 dakika boyunca santrifüj tüplerihücre enkazı. </li><li> 100,000 x g&#39;de 70 dakika süreyle ultrasantrifüj tüplerde süpernatantlar ve santrifüj toplayın. süpernatantlar atın. </li><li> Taze PBS 5 ml eksozom zengin pelet yeniden süspanse edin. 100,000 x g&#39;de 70 dakika boyunca tekrar taze ultrasantrifüj tüpleri ve santrifüj çözüm aktarın. </li><li> Sil süpernatantlar. eksozomlar uzun vadeli saklamak için, -80 ° C de PBS ve mağaza 50 ul pelet tekrar süspansiyon. Aşağıda gösterildiği gibi, alternatif olarak, bir protein ekstre doğrudan devam edin. </li></ol> 3. Protein Ekstraksiyon ve Hazırlık <ol><li> katma proteaz inhibitörü kokteyl ile 100-200 ul RIPA lizis ve ekstraksiyon tamponu izole exosomal pelet çözülür. Tampon pH 7.6, 25 mM Tris-hidroklorür, 150 mM sodyum klorid,% 1 NP-40,% 1 sodyum deoksikolat ve% 0.1 SDS (sodyum dodesil sülfat) oluşmaktadır. proteaz inhibitörü kokteyl Aprotinin, bestatin, l gibi proteaz inhibitörleri, çeşitli içermelidireupeptin, A ve EDTA (etilendiamin tetraasetik asit) pepstatin, bu proteazların bir dizi inhibe edebilir. </li><li> 13.000 xg (4 ° C) &#39;de 10 dakika boyunca eritildi çözüm santrifüj ve yeni bir 1.5 ml mikrosantrifüj tüplerine temizlenir süpernatantlar aktarın. </li><li> Bisinkoninik asit (BCA) ya da Bradford tahlili 16 kullanılarak eksozom Her numunenin protein konsantrasyonu ölçmek. </li><li> etiketlenmiş ve etiketlenmemiş örneklerden protein eşit miktarda (2 ug) karıştırın ve 120 mA&#39;da% 4-20 SDS-PAGE jel üzerinde eşit bir karışımı çalıştırmak / 200 V, 30 dakika. </li><li> Coomassie Mavisi ile Leke jel 17 lekenin izledi. </li><li> Bir jilet kullanarak, jel örnek şeritten kesti. Sonra 10-15 eşit parçaya jel şeritten kesti. 10-15 tüpler toplam taze bir 1.5 ml mikrosantrifüj tüp içine her parçası koyun. </li><li> % 50 asetonitril, girdap, 25 mM NH4 HCO3 küpleri daldırın ve süpernatantlar atın. İki kez tekrarlayın. KuruBir vakum konsantratör 18 19 küp. </li><li> 10 mM ditiyotreitol (DTT), girdap ve santrifüj kısaca ile küpleri rehidrate. 1 saat boyunca 56 ° C&#39;de inkübe edin. Süpernatantı atın. </li><li> 55 mM iyodoasetamit, girdap ve spin jel küpleri daldırın. 45 dakika boyunca, karanlıkta, oda sıcaklığında inkübe edin. Süpernatantı atın. </li><li> 25 mM NH 4 HCO 3, girdap, spin küpler daldırın ve supernatant atın. </li><li> % 50 asetonitril, girdap ve spin 25 mM NH 4 HCO 3 küpler daldırın. 3.9 ve 3.10 tekrarlayın. </li><li> bir vakum deriştirme aygıtı içinde, küpler kurutun. 25 mM NH4 HCO3 25 ul sıralaması derecesi modifiye tripsin ekleyin, 30 dakika boyunca 4 ° C&#39;de inkübe ve fazla solüsyonu atın. Tripsin olmayan 25 mM NH4 HCO3 küpleri bırakın ve gece boyunca 37 ° C enkübe edilir. </li><li> Kısaca aşağı küpler spin ve ekstra ortaya çıkan peptid transferiyeni bir tüpe CT. 30 dakika ve dönüş için, küp% 50 asetonitril içinde bir girdap% 5 formik asit içinde 30 ul ekle. ekstraktı ile süpernatant birleştirin. en az 5 ul bir vakum konsantratör peptid kurutun. </li><li> LC-MS / MS analizi için kütle spektrometresi çekirdek imkan numuneleri gönder. açık kaynak yazılım elde edilebilir MS analizi verileri, tipik bir katılım tanımlanan her protein için sayı, etiketli / etiketsiz oranları ve belirlenen eşsiz peptidler numarasını içerir. </li></ol> 4. Western Blot Doğrulama Not: Western lekeleme kütle spektrometresi sonuçları doğrulamak için tavsiye edilir. <ol><li> Standart batı lekeleme protokolleri takip ve yüklenen her gruptan protein, eşit miktarda sağlamak. MS tarafından belirlenen faiz proteinlerine karşı antikorlar (örneğin anneksin A5, laktat dehidrogenaz B zinciri) kullanın. dansitometrisi analysi ile Western lekeleme algılamas, MS protein tanımlama ve miktar doğru olduğunu teyit edebilir. </li></ol> 5. Proteomik Veri Analizi NOT: Her MS çoğaltmak için önemli bir protein adayların veri kalitesinin değerlendirilmesi, veri hazırlama, hesaplama ve kararlılık ayrı ayrı yapılır. Yukarıdaki analizler tamamlandığında, çoğaltır analiz verileri karşılaştırılmış ve 21 20, 6 birleştirdi. <ol><li> MS verilerin kalitesini değerlendirin. <ol><li> İlk olarak, SILAC-MS bir elektronik tablo programı / sayısal proteinlerin etiketsiz oranları etiketli dönüşümü log2. </li><li> bilimsel grafik ve istatistiksel yazılım, grup içinde 40-100 oran bidonları içine oranları ve bir histogram oluşturmak için göz başına oranları sayısını arsa. Histogram bir normal dağılım MS verilerinin 6 kaliteli olduğunu gösterir. Tüm MS çoğaltır için bu adımı yapmak. </li></ol></li><li>MS peptid oranları güven ve doğruluğunu artırmak için, en az iki sayısallaştırılmış peptidler olan proteinlerin kaldırmayı düşünün. </li><li> Önemli ölçüde yukarı ve aşağı düzenlenmiş protein adayları belirlemek için, önemi 22 eşikleri hesaplamak için aşağıdaki adımları kullanın. <ol><li> bilimsel grafik ve istatistik yazılımı olarak, frekans dağılım verilerine olmayan bir lineer regresyon veya eğri-fit oluşturmak. Bu adım, kesme değerlerini hesaplamak için kullanılabilir değerleri elde edilir. % 99 güven sınırları için 1.96,% 95 güven sınırları için σ veya 2.56 σ ± medyan olarak kesme değerlerini hesaplayın. NOT: kesilecek hesaplanması spesifik ayrıntılar Emmott ark bulunabilir. 22. </li><li> ya medyan altında (belirgin aşırı ifade edilir) ortanca üzerinde fazla 1.96 (veya 2.56) standart sapmalar veya daha düşük 1.96 (veya 2.56) standart sapmalar (kimin oranları protein adayları seçmekönemli ölçüde altında ifade edilir). </li></ol></li><li> tutarlılığı sağlamak için yukarıda tanımlanan önemli adayların çoğaltır karşılaştırın. Onlar bu son seçim adımı geçmesi için aşağıdaki kriterlere uygun olduğundan emin olun: Adayların sürekli tüm çoğaltır tespit edilmelidir; ve bir adayın çoğaltır düzenleme onların yönünde tutarlı olmalıdır (up-regüle veya aşağı-regüle tercihen bir adayın 3 çoğaltır üzerinden en az 2 ya da olmalıdır). </li><li> onların çoğaltır &#39;verilerini birleştirerek yukarıda belirtilen ölçütleri karşılayan adaylar için veri tamamlayın. </li></ol> 6. Biyoinformatik Doğrulama ve Karakterizasyonu NOT: Mevcut genomik ve biyoinformatik bilgiler hemen hemen her protein için bilgi hazinesi sunuyor. Bu bilgilere Veri madenciliği ve biyoinformatik analizi önemli adayların mülkiyet ve işlevleri içgörü büyük bir kazanma konusunda yardımcı olabilir. bu Process uygun aşağı ıslak laboratuvar deneyleri tasarlamak için genellikle gereklidir. <ol><li> Onların GenBank Erişim numaraları veya UniProt kimlikleri, mevcut eksozom veritabanlarının karşı aday arama (Exocarta 23, Evpedia 24) kullanarak aday proteinleri daha önce exosome bulunmuştur doğrulamak için. Bu adım adayları Eksozomlann gerçekten de olan güven bir katman daha ekler. <ol><li> Erişim http://www.exocarta.org/, &quot;sorgu&quot; ve giriş ya gen veya protein adı / erişim numarası tıklayın. Bir özet sayfası görünecek ve exosome deliller herhangi varsa sağlanan gösterilir. </li></ol></li><li> HIV-1 ve insan Protein Etkileşimi Veri 25 aleyhinde bulundu. Alternatif olarak, aday proteinlerin etkileşim ve deney durumu hakkında bilgi verebilir belirli veritabanlarında arama. <ol><li> www.ncbi.nlm.nih.gov/RefSeq/HIV de veritabanına erişmekEtkileşimler. &#39;Protein etki alanı adı&#39; Girişte adayların adlarını veya katılım numaralarını girin ve arama tıklayın. Arama sonuçları HIV-1 ve protein adaylar arasındaki etkileşimlerin içgörü sağlamak ve gerçekten HIV-1 ile ilişkili olabilir adayların hangi önerebilirsiniz. </li></ol></li><li> İthalat GenBank numaraları ya da (örneğin FunRich, Fonksiyonel Zenginleştirme analiz aracı 26 gibi) GO analiz yazılımı içine adayların UniProt kimlikleri ve yazılımı çalıştırmak. <ol><li> http://www.funrich.org/ de yazılımını indirin. Yükleme tamamlandıktan sonra, zenginleştirme analizi altında, tıklayın &quot;Veri Kümesi Ekle&quot; Yazılımı açmak ve protein / genlerin listesini yükleyin. Sonraki GO analizi görselleştirmek için grafik türünü seçin. NOT: GO analiz sonuçları pasta grafikler şeklinde görüntülenmiştir edilecektir. Bu adım (Biyolojik Süreci (BP), Hücresel Bileşeni alanlarında adayları ile ilişkili küresel fikir edinmek için C yardımcı olurC) ve moleküler işlev (MF). </li></ol></li><li> Erişim DAVID http://david.ncifcrf.gov/ 27 de, kendi web sitesinde &quot;Fonksiyonel Annotation Tool&quot; tıklayın. Gibi &quot;UniProt kimliği&quot; olarak genin &quot;Identifier&quot; seçeneğini, aday listesine girin &quot;Gen Listesi&quot; ve arama seçin. zenginleştirilmiş GO şartları, p-değerleri ve diğer parametreler &quot;Gene_Ontology&quot; kategorisi altında &quot;Annotation Özeti Sonuçları&quot; sayfasını tıklayarak ulaşabilirsiniz. </li><li> Diğer proteinler ile adayların potansiyel etkileşimleri araştırmak için, potansiyel protein-protein etkileşimleri ve olası biyokimyasal yollar aydınlatmak için açıkça erişilebilir STRING veritabanı 28 kullanın. NOT: Analiz ve Fonksiyonel Açıklaması (Bölüm 6,3-6,4) GO da son STRING yazılımı kullanılarak yapılabilir. <ol><li> http://string-db.org/ de veritabanına erişmek. belirlenen s girdi protein kimliği veya diziAraması kutu ve analiz için doğru tür seçin. &quot;Ara&quot; düğmesini tıklatın. Sonuçlar hem doğrudan (fiziksel) ve dolaylı (işlevsel) dernekler hakkında bilgi verecektir. exosomal aday için ilk on bilinen maçlar görüntülenir ve önemli aday seçiminde dikkate alınmalıdır. NOT: Adayların ile ilişkili bilgilerin bir hayli yukarıdaki adımları kullanarak analiz edilebilir. en önemli adaylar, daha aşağıda moleküler ve biyokimyasal analiz için seçilebilir. </li></ol></li></ol>

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T., Lempicki, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nat Protoc. 4 (1), 44-57 (2009).</li><li>Szklarczyk, 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=The+STRING+database+in+2011:+functional+interaction+networks+of+proteins,+globally+integrated+and+scored.">The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored.</a> Nucleic Acids Res. 39, 561-568 (2011).</li><li>Thery, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Protoc Cell Biol. , (2006).</li><li>Lobb, R. 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=Optimized+exosome+isolation+protocol+for+cell+culture+supernatant+and+human+plasma.">Optimized exosome isolation protocol for cell culture supernatant and human plasma.</a> J Extracell Vesicles. 4, 27031 (2015).</li><li>Webber, J., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+pure+are+your+vesicles.">How pure are your vesicles.</a> J Extracell Vesicles. 2, (2013).</li><li>Cantin, R., Diou, J., Belanger, D., Tremblay, A. M., Gilbert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+between+exosomes+and+HIV-1:+purification+of+both+vesicles+from+cell-free+supernatants.">Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants.</a> J Immunol Methods. 338 (1-2), 21-30 (2008).</li><li>Chertova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+and+biochemical+analysis+of+purified+human+immunodeficiency+virus+type+1+produced+from+infected+monocyte-derived+macrophages.">Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages.</a> J Virol. 80 (18), 9039-9052 (2006).</li><li>Nikolov, M., Schmidt, C., Urlaub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry-based+proteomics:+an+overview.">Quantitative mass spectrometry-based proteomics: an overview.</a> Methods Mol Biol. 893, 85-100 (2012).</li></ol>

The authors have declared no conflict of interest.

Bu yazıda tarif edilen prosedürlere olarak, ana exosomal proteomu HIV-1 enfeksiyonunun etkisini araştırmak için SILAC tekniğin uygulanmasını göstermektedir. Başlangıçta, enfekte edilmemiş ve HIV-1 ile enfekte edilmiş hücreleri, farklı olarak, izotopla işaretli bulunmaktadır. farklı şekilde etiketlenmiş eksozomlar sonra protein çıkarma yapmadan önce saflaştırılır. Daha sonra, sıvı kromatografi-tandem kütle spektrometrisi exosomal Proteomun analiz için kullanılır. Son olarak, elde edilen ...

viral enfeksiyonlar da dahil olmak üzere bir çok insan hastalıkları, sık sık etkilenen hücrelerin etrafında yer alan farklı hücresel süreçleri ile ilişkilidir. Proteinler, genellikle bu süreçleri arabuluculuk, nihai hücresel efektör olarak hareket. Proteinlerin analizi sıklıkla etkilenen hücrelerin yerel çevreye kadar çok değerli bilgiler sağlar ve hastalık patogenezinde altta yatan mekanizma anlamak için bize yardımcı olabilir. Çeşitli protein analiz teknikleri arasında, proteomik özellikle büyük umut vaat ediyor. güçlü, geniş çaplı bir aracı olarak, proteomik, özellikle fonksiyon ve protein etkileşim alanında, hücresel süreçlerin bir tarihsel sağlayabilir. Belirli proteinlerin analiz İnceleme bölgesinde, araştırmacılar, hücresel bileşenler, özellikle de proteinlerinin ekspresyonunu izlemek için izin etiketleme teknikleri geliştirilmesi yoluyla basit yapılır. Birçok proteomik analizler hücresel gerçekleştirilen olmasına rağmenproteom ölçeği, hücre içi bölmeleri üzerinde proteomik karakterizasyonu 1 özellikle bilgilendirici olduğu kanıtlanmıştır. Bu HIV-1 enfeksiyonu ile ilgili çalışmalarda iyi örneklenmiştir. Eksozomlar, hücre tipleri 2, 3, geniş bir yelpazede tarafından salgılanan 30-100 nm zar vezikülleri arası iletişim ve moleküler taşıma önemli bileşenleridir. Daha önce HIV-1 tomurcuklanma 4, 5 önemli roller oynamaya keşfedildi. Fonksiyonel diseksiyon ile proteomik analizi birleştirerek, HIV-1 ile enfekte hücrelerden salınan eksozomlar hücresel apoptoz ve çoğalması 6 olmak üzere alıcı hücreler, komşu hücresel özelliklerini etkileyecek eşsiz ve nicelik farklı protein imza ve liman düzenleyici moleküller oluşur bulundu. yöntemler bu protokolde tarif edilmiştir,yani SILAC HIV-1 enfekte hücreleri eksozom 7 göre proteomik karakterizasyonu (hücre kültürü içinde amino asitler ile izotop işaretleme). Benzer yaklaşımlar, daha özel bir bölme veya ilgi fraksiyonuna deneysel gerilme ayarlama ve tarif edilen prosedürlere, gerekli değişiklikler yaparak patojenez sırasında, diğer alt-hücresel bölümleri anlamak için uygulanabilir. kantitatif proteomik yöntemlerin son gelişmeleri göz önüne alındığında, belirli bir deney için en etkili yöntem seçerken seçim için pek çok vardır. Bunlar arasında kimyasal bazlı iTRAQ (göreli ve mutlak ölçümü için izobarik etiketleri) 8 ve etiket içermeyen MRM (çoklu reaksiyon izleme) 9 teknikleridir. Her iki yöntem güçlü araçlardır ve özel ayarlar için iyi seçimlerdir. Tipik bir laboratuvar ağırlıklı hücre hatları ile çalışmak için, ancak, bu iki yöntem şansı göreceli vary yüksek maliyetler ve vardır daha zaman alıcı SILAC tabanlı yönteme göre. SILAC hücresel protein olarak kültür ortamından amino asit radyoaktif olmayan izotop formlarını içeren bir metabolik göre etiketleme tekniği. Tipik haliyle, SILAC deneyler, örneğin iki hücre popülasyonlarının, enfekte olan ve olmayan ile başlar. Tam etiketleme elde edilene kadar her diferansiyel kendine özgü izotopik ortamda etiketlenir. Bu hücrelerin etiketli eksozomlar sonra protein ekstre etmeye tabi tutulmaktadır. Bir kez etiketli exosomal proteinleri sıvı kromatografisi tandem kütle spektroskopisi 10 kullanılarak analiz edilmektedir, ayıklanır. Son olarak, kütle spektrometresi sonuçları ve önemli ölçüde etiketli proteinler istatistiksel tabi tutulur ve biyoinformatik titiz biyokimyasal doğrulama yanı sıra analiz eder. Bizim daha önceki soruşturma raporları hücre hatları bir genellikle olduğu gibi SILAC / eksozom prosedürleri, birincil hücrelerden daha hücre hatları için daha uygun olacağı kanısındayızverimli izotop etiketleme için aktif prolifere devlet,

<table><tbody><tr><td>H9 cell line</td><td>ATCC</td><td>HTB-176</td><td></td></tr><tr><td>Trypan Blue</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>MTT assay kit</td><td>Thermo Fisher</td><td>V13154</td><td></td></tr><tr><td>Dialyzed fetal bovine serum (FBS)</td><td>Thermo Fisher</td><td>26400044</td><td></td></tr><tr><td>SILAC Protein Quantitation Kit &amp;ndash; RPMI 1640</td><td>Thermo Fisher</td><td>89982</td><td>DMEM version (89983)</td></tr><tr><td>L-Arginine-HCl, &lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;, &lt;sup&gt;15&lt;/sup&gt;N&lt;sub&gt;4&lt;/sub&gt; for SILAC</td><td>Thermo Fisher</td><td>88434</td><td></td></tr><tr><td>L-Lysine-2HCl, &lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt; for SILAC</td><td>Thermo Fisher</td><td>88431</td><td></td></tr><tr><td>HIV-1 NL4-3</td><td>NIH AIDS Reagent Program</td><td>2480</td><td></td></tr><tr><td>Alliance HIV-1 p24 Antigen ELISA kit</td><td>PerkinElmer</td><td>NEK050001KT</td><td></td></tr><tr><td>Refrigerated super-speed centrifuge</td><td>Eppendorf</td><td>22628045</td><td></td></tr><tr><td>Refrigerated ultracentrifuge</td><td>Beckman Coulter</td><td>363118</td><td>Should be able to reach 100,000 x g</td></tr><tr><td>50 mL Conical Centrifuge Tubes</td><td>Thermo Fisher</td><td>14-432-22</td><td></td></tr><tr><td>Ultracentrifuge Tubes</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>SW 32 Ti Rotor</td><td>Beckman Coulter</td><td>369694</td><td></td></tr><tr><td>RIPA buffer</td><td>Thermo Fisher</td><td>89900</td><td></td></tr><tr><td>Protease Inhibitor Cocktails</td><td>Thermo Fisher</td><td>&amp;nbsp;78430</td><td></td></tr><tr><td>ThermoMixer&amp;nbsp;</td><td>Eppendorf</td><td>5384000020</td><td></td></tr><tr><td>BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Fisher</td><td>23250</td><td></td></tr><tr><td>Spectrophotometer</td><td>Biorad</td><td>1702525</td><td></td></tr><tr><td>SDS PAGE Gel apparatus</td><td>Thermo Fisher</td><td>EI0001</td><td></td></tr><tr><td>Novex 4-20% Tris-Glycine Mini Gels</td><td>Novex</td><td>XV04200PK20</td><td></td></tr><tr><td>Gel staining reagent</td><td>Sigma Aldrich</td><td>G1041</td><td></td></tr><tr><td>Sequencing Grade Modified Trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>SpeedVac Concentrator</td><td>Thermo Fisher</td><td>SPD131DDA</td><td></td></tr><tr><td>Antibody to human annexin A5</td><td>Abcam</td><td>ab14196</td><td></td></tr><tr><td>Antibody to human lactate dehydrogenase B chain</td><td>Abcam</td><td>ab53292</td><td></td></tr><tr><td>Graphing and Statistical Software</td><td>Systat&amp;nbsp;</td><td>SigmaPlot&amp;nbsp;</td><td>Or GraphPad Prism</td></tr><tr><td>Quantitative proteomics software suite</td><td>Max Planck Institue of Biochemistry</td><td>Maxquant&amp;nbsp;</td><td></td></tr><tr><td>Software and databases</td><td>Various vendors</td><td></td><td>Refer to main text for details</td></tr></tbody></table>

silac based proteomic characterization of exosomes from hiv-1 infected cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Şekil 1A SILAC etiketleme prosedürü 21 gösteren bir akış şemasıdır. eksozomlar arındırmak için, numuneler santrifüj yoluyla döndürülür gerekir. Şekil 1B seri Ultrasantrifügasyon 21 exosome saflaştırma adımları göstermektedir. saflaştınldı sonra prosedür de tarif edilen, eksozomlar deney proteomik analize tabi tutulur. ...

Watch this Scientific Journal Video about SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells at JoVE.com

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Burada, ana exosomal proteomlarda HIV-1 enfeksiyonunun etkilerini analiz etmek için hücre kültürü (SILAC) amino asitler ile izotop etiketleme tekniği kullanılarak kantitatif proteomik bir yöntem açıklanmaktadır. Bu protokol, kolayca farklı gerilme veya enfeksiyon koşullarda hücrelere uyarlanabilir.

HIV-1 enfekte olmuş hücrelerden eksozom SILAC Bazlı proteomik Karakterizasyon

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

silac-based-proteomic-characterization-exosomes-from-hiv-1-infected

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

10.6K Görüntüleme.  Brown University.  Burada, ana exosomal proteomlarda HIV-1 enfeksiyonunun etkilerini analiz etmek için hücre kültürü (SILAC) amino asitler ile izotop etiketleme tekniği kullanılarak kantitatif proteomik bir yöntem açıklanmaktadır. Bu protokol, kolayca farklı gerilme veya enfeksiyon koşullarda hücrelere uyarlanabilir. 

Video: SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Kantitatif Kütle Spektrometresi Analiz lipid Damlacık izolasyonu

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Biyokimya

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Görüntülemede HIV-1 Zarf bağlı Virolojik Synapse ve Sentetik Lipid Bilayers üzerine Sinyal

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

İmmünoloji ve Enfeksiyon

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Fonksiyonel Motifler ve Bağlama Ortaklar peptid bazlı Kimlik

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

Bir Hücre temelli bir ELISA testi kullanarak, HIV-1 zarf trimerik Glikoproteinler konformasyonel Değerlendirilmesi

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

HIV-1 pozitif Bireylerin Plazma eksozomlann izolasyonu

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA İçeren Exosomes İzolasyonu ve Karakterizasyonu

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Biyoloji

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Tek hücreli Nefelometri mRNA ve yüzey Protein Simian virüs, immün yetmezlik enfekte CD4 ifadede+ T hücreleri izole Rhesus makak üzerinden

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

Yüksek Verim Ekstrasellüler Vezikül Preparatlarının Virüsten Uzaklaştırılması

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

Multiparametre Optofluidik Platform Kullanılarak Kalsiyum Akını ve HIV-1 Enfeksiyonunun Tek Hücreli Karakterizasyonu

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

SILAC-immunoprecipitation Kantitatif PROTEOMİK kullanma Memeli Hücrelerde Protein Etkileşim Ortakların Kimlik

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...