Name: silac based proteomic characterization of exosomes from hiv-1 infected cells
Uploaded: 2017-03-03T12:30:00
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, kullanılabi...

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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,

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		<tr height="21">
			<td height="21" style="height:21px;width:227px;">H9 cell line</td>
			<td style="width:121px;">ATCC</td>
			<td style="width:111px;">HTB-176</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Trypan Blue</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">15250061</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">MTT assay kit</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">V13154</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:227px;">Dialyzed fetal bovine serum (FBS)</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">26400044</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">SILAC Protein Quantitation Kit &#8211; RPMI 1640</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">89982</td>
			<td style="width:168px;">DMEM version (89983)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">L-Arginine-HCl, 13C6, 15N4 for SILAC</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">88434</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">L-Lysine-2HCl, 13C6 for SILAC</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">88431</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">HIV-1NL4-3&#160;&#160;</td>
			<td style="width:121px;">NIH AIDS Reagent Program</td>
			<td style="width:111px;">2480</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Alliance HIV-1 p24 Antigen ELISA kit</td>
			<td style="width:121px;">PerkinElmer</td>
			<td style="width:111px;">NEK050001KT</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Refrigerated super-speed centrifuge</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:111px;">22628045</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Refrigerated ultracentrifuge</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">363118</td>
			<td style="width:168px;">Should be able to reach 100,000g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">50mL Conical Centrifuge Tubes</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">14-432-22</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ultracentrifuge Tubes</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">326823</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SW 32 Ti Rotor</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">369694</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">RIPA buffer</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">89900</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Protease Inhibitor Cocktails</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">&#160;78430</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">ThermoMixer&#160;</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:111px;">5384000020</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">BCA Protein Assay Kit&#160;</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">23250</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Spectrophotometer</td>
			<td style="width:121px;">Biorad</td>
			<td style="width:111px;">1702525</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SDS PAGE Gel apparatus</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">EI0001</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Novex 4-20% Tris-Glycine Mini Gels</td>
			<td style="width:121px;">Novex</td>
			<td style="width:111px;">XV04200PK20</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Gel staining reagent</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:111px;">G1041</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sequencing Grade Modified Trypsin</td>
			<td style="width:121px;">Promega</td>
			<td style="width:111px;">V5111</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SpeedVac Concentrator</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">SPD131DDA</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Antibody to human annexin A5</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:111px;">ab14196</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Antibody to human lactate dehydrogenase B chain</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:111px;">ab53292</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:227px;">Graphing and Statistical Software</td>
			<td style="width:121px;">Systat&#160;</td>
			<td style="width:111px;">SigmaPlot&#160;</td>
			<td style="width:168px;">Or GraphPad Prism</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Quantitative proteomics software suite</td>
			<td style="width:121px;">Max Planck Institue of Biochemistry</td>
			<td style="width:111px;">Maxquant&#160;</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Software and databases</td>
			<td style="width:121px;">Various vendors</td>
			<td style="width:111px;"></td>
			<td style="width:168px;">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 ...

Research

JoVE Journal

Immunology and Infection

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Yeşil Floresan Protein ifade Rev-bağımlı lentiviral Vector kullanarak HIV-1 Olumlu Hücreler Özgül İşareti

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

HIV-1 tropizm genotipik Çıkarım kullanarak Nüfus tabanlı Sıralama V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Hazırlanması ve kullanımı, HIV-1 Enfekte İlköğretim CD4 + T-Hücreleri Hedef Hücreler olarak Natural Killer Hücre Sitotoksik Tahliller

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Standart Klinik Tahliller Algılama Sınırı Aşağıda Viral Yükler HIV enfekte kişilerde kuvvetlendirme ve HIV-1 RNA miktarının

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

HIV-1 Çekirdek in vitro Uncoating

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

Yeni Araçlar HIV-1 ile enfekte bireyler gelen Düzenleyici T hücreleri büyütmek için

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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

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

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. ...