Name: proteomic profiling of macrophages by 2d electrophoresis
Uploaded: 2014-11-04T15:00:00
Description: Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).

Protokolü kurumların insan araştırma etik kurulunun yönergeleri takip eder. Sağlıklı insan donörlerden Buffy coat Bölge Kan Transfüzyon Merkezi (Lille, Fransa) elde edilmiştir. Buffy kat elde edilen Numune bir Inserm koleksiyon (n ° DC2010-1209) olarak ilan edilmiştir. 1. Malzeme ve Kültür Medya Hazırlık <ol><li> 1x PBS elde etmek için steril damıtılmış su içinde, 10X fosfat tamponlu salin (PBS) ile seyreltilir. </li><li> Ve% 10 toplanmış insan serumu olmadan gent...

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Bu tarifnamede tarif edilen protokol makrofajlar, M1 (pro-enflamatuar) ve M2 (anti-iltihap) olarak iki alt-tip farklı uyaranların etkisini analiz etmek için bir yöntem göstermektedir. Daha önce 7 yayınlanan M1 ve M2 makrofajların birincil kültürleri monositlerin farklılaşması elde edilmiştir. 2D DIGE jel elektroforezi prosedür, Cy3 ve Cy5 floresans bir tarayıcı için uyarım / emisyon dalga iki kez jel tarama yapmak için SDS-PAGE için izoelektro-odaklanma, düş...

Makrofajlar, ayırt edici fonksiyonel fenotipleri elde edebiliyoruz heterojen ve plastik hücrelerdir. İn vivo olarak, bu hücreler mikrobiyal ürünler, sitokinler, vb. 1 mikro çevre signalssuch büyük bir çeşitlilik yanıt. İn vitro, pro-enflamatuar fenotip (M + -1) makrofaj lipopolisakarit (LPS) ve interlökin-4 (IL-4) gibi sitokinler tarafından anti-iltihabik bir fenotipe (M2) tarafından indüklenebilir. Dahası, makrofajlar belirli sinyalleri 2 üzerine, tersine M2 fenotip bir aktive M1 geçiş yapabilirsiniz. Fenotip bağlı olarak, makrofajlar farklı işlevlere sahip olacaktır. M1 makrofajlar mikroorganizmaları ve tümör hücrelerini öldürmeye 3, tümör nekroz faktörü α (TNF-α) gibi pro-enflamatuar sitokinleri üreten hücrelerdir. Buna karşılık, M2 makrofajlar anti-enflamasyon üreterek yara iyileşmesi ve fibrozis gibi bu enflamatuar yanıtı önlemekörneğin 3,4, TGF-β Y faktörler. Daha önce Boyum 6 uyarlanan bir teknik kullanarak 5 tarif edilen sağlıklı donörlerden elde edilen insan periferal kan tek-çekirdekli hücreleri Ficoll yoğunluk farklı santrifüjü yoluyla izole edilmiştir. Kültür makrofajlar primer kültür 7 6 gün sonra M1 veya M2 fenotip ayırt edilebilir. Protein ekspresyonu ya da ana bilgisayar patojenik veya mikrobik toksinler gibi çeşitli kontrollü uyaranlarla altında makrofajların iki alt tip arasında, protein değişikliklerin analizi, pro- ve anti-inflamatuar makrofaj özelliğe deşifre yararlı olacaktır. Proteomiks özellikle yukarı veya çeşitli uyaranlara altında insan kültürlü makrofajlar aşağı-regüle olan proteinlerin doğrudan izlenmesi için benzersiz araçlardır. Floresan boyalar gibi düşük duyarlılık ve görüntü analizi 8 gibi 2D jel elektroforezi sınırlamaları, bazı çözdük &lt;<html> / Sup&gt;. Sistein kalıntıları ile reaksiyona sokulması boyalar lizin kalıntıları 9 ile reaksiyona kıyasla tespit karşı artmış bir hassasiyet. Önceki bir çalışmada, klasik, gümüş-lekeli 2D elektroforez 11 kıyasla çok az örnekleri 10 analizinde DIGE doygunluk etiketleme yararını göstermiştir. Bu teknoloji hızlı bir makrofaj iki alt tip arasında veya aynı tipinin muamele edilmemiş ve edilmiş makrofajlar arası protein değişiklikleri analiz etmek yararlıdır. Bu proteomik tekniğin avantajları, 2 boyutlu jel 12 analiz protein boyut ve post-translasyonel modifikasyonları bilgilere erişim sahip olanlardır. Bu analiz edilebilir numunelerin sayısının sınırlandırılması, bu yüksek verimli bir yöntem değildir dikkate alınmalıdır. Kütle spektrometresi dayalı yüksek verimli deneyler gelişimi gözden olarak son 13 bu artırabilirsiniz. _content &quot;&gt; Burada elektroforez, izoelektro-odaklanma süreçleri yoluyla kültürlü makrofajlar protein çıkarılması 2D DIGE analizi gerçekleştirmek için nasıl sunmak ve yeterli 2D yazılım kullanışlılığı SDS-PAGE yanı sıra bilgi.

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			<td>GE Healthcare</td>
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			<td>GE Healthcare</td>
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			<td>T9281</td>
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			<td>Sigma-Aldrich</td>
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			<td height="17" style="height:17px;">electrode wicks</td>
			<td>Bio-Rad</td>
			<td>165-4071</td>
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proteomic profiling of macrophages by 2d electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Uygun diferansiyel proteomik analizi gerçekleştirmek için, analiz edilecek örneklerin işlenmesi doğrulanması gerekir. Önce 5 yayınlanan sunulan örnekte, makrofaj hücre kültürü kalitesi, morfolojik ve moleküler özellikler için gereklidir. Makrofajlar monositlerin farklılaşması ve kültürün homojenliği faz mikroskobu ile takip edilmiştir. Şekil 1, M1 ve M2 makrofajların primer kültürlerinde bir örnek göstermiştir. Gösterildiği gibi, ...

Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

Proteomic Profiling of Macrophages by 2D Electrophoresis

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

2D Elektroforez ile Makrofagların proteomik Profil

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

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