Yazarlar, bu yöntemin ilk olarak John Woolford kabul etmek istiyorum, TGK GM057483 ve AI076245 NIH tarafından desteklenen ve ÇHC GM77073 tarafından desteklenmektedir.

1. % 7-47 sakkaroz degradelerin hazırlanması <ol><li> Gradyanlar sürekli olmasına izin kullanmadan önce sakaroz gradyanlar bir gün hazırlayın. % 7 steril ve 4, 50mm NH 4 Cl içeren% 47 sakaroz çözümleri, 50mm Tris-asetat pH 7.0 ve 12mm MgCl 2 ve mağaza ° C 1 . </li><li> 6 gradyanlar aşağıdaki sakaroz konsantrasyonu her 48 ml için% 7 ve% 47 sukroz stok çözelti karışımı için. 1mm bir nihai konsantrasyonu her sakaroz çözeltisini DTT (1M stok) ekleyin. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Final Konsantrasyon </td><td> 7% sakaroz </td><td> 47% sakaroz </td></tr><tr><td> % 7 </td><td> 48 ml </td><td> 0 </td></tr><tr><td> % 17 </td><td> 36 ml </td><td> 12 mL </td></tr><tr><td> % 27 </td><td> 24 ml </td><td> 24 ml </td></tr><tr><td> % 37 </td><td> 12 mL </td><td> 36 ml </td></tr><tr><td> % 47 </td><td> 0 </td><td> 48 ml </td></tr></tbody></table></li><li> Gradyanlar dökmek için, 20 ml şırınga Parafilm ile güvence altına alınması ve sızdırmazlık Pasteur pipeti eklemek veya uzun bir iğne kullanınız. 1 x 3.5 &quot;polyallomer ultrasantrifüjdeki tüp alt% 7 sakaroz 7 mL ekleyin. Sonraki 7 mL, 0.3 &#39;ün, tüpün alt kısmına yakın Pasteur pipet yerleştirerek ve herhangi bir hızlı pipetleme altında% 17% 7 çözüm sakaroz çözeltisini eklemek 7 mL% 27,% 37 ve% 47 sukroz çözümleri ile ml / sn. tekrarlayın. karıştırma minimum 4 ° C gecede rotorlar, şişe ile birlikte. Mağaza gradyanlar meydana geldiğini belirterek, katmanları arasında net çizgiler dikkat etmelisiniz örnekleri hasat için kullanılacak tüpleri. </li></ol> 2. Maya özü hazırlanması <ol><li> 0.8-1.0 bir OD 600 125 ml maya kültürü büyütün. Nihai konsantrasyonu 100 mg / ml kültür Siklohekzimidsiz ekleyin ve 30 sallayarak ° C&#39;de 15 dakika süreyle devam. </li><li> Bu arada, 80 mikrogram / ​​ml sikloheksimid, 200 mg / ml heparin,% 0.2 DEPC, ve 10 mM Tris-HCl, pH 7.5, 0.1M NaCl, 30mm MgCl 2 içeren lizis tamponu hazırlamak. Bu noktadan itibaren buz her şeyi tutun. </li><li> 250 ml santrifüj şişeye kültür dökün ve buz ile doldurun. 5 dakika 4 ° C için 8.000 xg (SLA-1500 rotor 7250 rpm) Santrifüj Pelet buz lizis tamponu ve 15 ml polipropilen tüplere transfer 10 ml ile bir kez yıkayın. 3 dakika 4 ° C 5900 xg (SLA-600TC rotor ~ 6,000 rpm) Santrifüj 1.5mL tüp lizis tamponu, transfer 0,5 mL pelletini tekrar ve soğutulmuş, pişmiş asitle yıkanmış cam boncuklar 0.5 mL ekleyin. Vorteks, 30 sn arasında her aralık buz tüpü soğutma dört 30 sn aralıklarla hücreleri. Lizis tamponu başka bir 0.5 ml 1.5 ml tüp içine ekleyin. Santrifüj bir mikrosantrifüj maksimum hızı (bir Eppendorf 5417R mikrosantrifüj 14.000 rpm) 10 dk 4 ° C Supernatant yeni bir 1.5 mL mikrosantrifüj tüpüne aktarın. OD 260 / OD 280 oranı (bkz. aşağıda adım 3.1) belirlemek için ayrı bir tüp 10 ul çıkarın. -80 Mağaza özleri ° C </li></ol> 3. Memeli hücrelerinde alıntılar hazırlanması <ol><li> Yapışık hücreler için ~% 70 izdiham 100 mm tabak içinde büyür. 11 ml degrade başına bir 100 mm çanak (tipik verim ~ 20 OD 260 adet) ihtiyacınız olacak. Doğrusal degrade boyutları daha büyük veya daha küçük miktarda Ölçeği. Yapışmaz hücreleri için, 11 ml degrade başına yaklaşık 1 x 10 7 hücreleri ihtiyaç vardır. </li><li> Lizis için, önce 100 mcg / mL Siklohekzimidsiz ekleyin. 37 ° C&#39;de 15 dakika inkübe edin. Buz plakaları aktarın ve hücreleri buz PBS içinde iki kez yıkayın. Gelecekteki tüm adımları 0-4 ° C&#39;de yapılan ZORUNLU </li><li> Aspirasyon PBS tüm izlerini silmek (plakalar buz açılı yatakta süzdürülür) ve 1 ml lizis tamponu, kazıma ve önceden soğutulmuş 1,5 mL tüp transfer ekleyin. Lizis tampon parçaları, hücre tipi ve büyüme koşulları için optimize edilmiş olması gerekir. Iyi bir başlangıç ​​tampon 20 mM Hepes KOH, pH 7.4, 15 mM MgCl 2, 200 mM KCl,% 1 Triton X-100 (v / v), 100 mg / ml sikloheksimid, 2 mM DTT, 1 mg / ml heparin. Temel değişkenler, magnezyum ve potasyum klorür gibi dahil edilmesi veya çıkarılması sitoskeletal veya membran bağlı polysomes artırmak için non-iyonik deterjan dışlanma konsantrasyonları. 27 gauge şırınga iğnesinin iki kez geçerek ya da B Tipi havaneli kullanarak Dounce homojenizasyon 5-10 darbeleri hücreleri Lyse. Şırınga veya Homojenizatör kullanmadan önce ön soğutulmuş olmalıdır. </li><li> 5 dakika buzdolabında mikrosantrifüj 14.000 xg&#39;de dönerler. Sukroz gradient (memeli hücreleri için bu Triton X-100 ve heparin eksik lizis tamponu yapılır ama başka türlü, 1.2 ve 1.3 &#39;de açıklandığı gibi) ya da daha sonra kullanılmak üzere sıvı azot flaş dondurma taze lizat aktarın. </li></ol&gt; 4. Gradyanlar santrifüj <ol><li> Buz üzerinde polyribosome lizatları çözülme. Ayrılmış 10 mcL örnek 1 ml su ekleyerek ve bir spektrofotometre ile OD 260 / OD 280 oranı okuma lizat nükleik asit konsantrasyonu belirleyin. Açıklandığı gibi yetişen bir maya kültürü tipik verim 50 OD adettir. </li><li> Degrade yüzeyine yakın tüp duvar, hafifçe degrade üstüne lizat 25 OD 260 adet tabaka karşı yerleştirilen ucu ile bir pipet kullanarak. Için lizatı 35 mL degrade 500 ul genellikle yüklenir. Lizis tamponu tüm gradyanlar eşit hacimde yüklü sağlamak için kullanılabilir. Düşük miktarda malzeme daha iyi çözünürlük verir. Forseps kullanarak, sallanan bir kova rotor kova içine dikkatlice düşük gradyanlar. </li><li> Xg 100.000 (Surespin 630 rotor 23.000 rpm), 4 saat 4 gradyanlar Santrifüj ° C Bu protokol, küçük hacimli gradyanlar için adapte edilebilir. </li></ol> 5. Veri ve kesirler Toplanması <ol><li> Yaklaşık 30 dakika önce tamamlanması için 4 saat spin Model 184 tüp delici iğne takın. Online Isco Model kalem takın UA-5 Absorbans / Floresan Monitör. Absorbans Güç istikrarlı bir temel analiz örnekleri önce ulaşılması izin izlemek için. Polysome profil Elektronik satın alma grafik kaydedici DI-148U veri toplama ünitesi (DATAQ Araçlar) takarak kolayca elde edilebilir. Profiller daha sonra gerçek zamanlı olarak toplanan ve beraberindeki WinDaq yazılım kullanarak daha sonra ek açıklama için kaydedilmiş olabilir. </li><li> Fluorinert ters akış, hızlı ayarını kullanarak 50ml şırınga pompası doldurun. Hiçbir hava kabarcığı olmadığından emin olun. Şırıngadan tüp delici hortumunu bağlayın ve kısaca, herhangi bir hava kabarcığı hortumu temizlemek için 3 ml / dk ileri yönde Fluorinert akışını açmak. </li><li> Örnek toplamak için akış hücresi sonunda hortumun altında kesirler toplama çalıştırırsanız, tüpler atık beher veya seri yerleştirin. </li><li> Sukroz gradient santrifüj rotor içeren polyallomer santrifüj tüplerine dikkatlice çıkarın ve buz üzerinde (rahatsız edici bir renk geçişlerini önlemek için boş bir tüp ile buz kuyu öncesi form) koyun. </li><li> Hücre özleri analiz etmek, tüp delici degrade tüpü yerleştirin. Tüpün üst prizine bağlayın ve güvenli. Iğne tüpün alt deler ve böylece alt aşamasında santrifüj tüpüne kaldırın. Sonra iğne santrifüj tüpüne dibinden dışarı fırlamıştı, 6mL/min ileri yönde Fluorinert akışı başlar. </li><li> Fluorinert tüp içine akan başladıktan sonra, online absorbans monitör iğne hareketini izlemek. İğnenin taşımak için 60 bir hız ayarı grafik hareketi açmak başlar. 40S ribozomal altbirim polyribosome kompleksleri boyunca devam algılama ve takip serbest malzeme ile başlayan monitör OD 254 kayıt edecektir. RNA veya protein analizi için kesirler toplama ise, tipik olarak 0.2 dakika kesirler (1 ml) alınır. RNA analizi için, kesirler ekstraksiyon tercih edilen yöntem bağlı olarak, doğrudan Trizol (Invitrogen), Fenol veya SDS / proteinaz K içeren tüp içine toplanan olmalıdır. </li><li> Polyribosome profil sonuna ulaşıldığında, Fluorinert, akış santrifüj tüpüne kapatın ve absorbans / floresan monitör grafik hareketi durdurmak. </li><li> Şırıngaya tüp sakaroz çekmek için emin olmak için hızlı bir oranında ters yönde akışını başında şırıngaya santrifüj tüpüne Fluorinert geri çekin. Santrifüj tüpüne sökün tüp delici iğne, alt ve tüp kaldırmak. </li><li> Her sukroz gradient için 5.8 üzerinden 5.5 adımları tekrarlayın. </li><li> Tüm sakaroz gradyanlar analiz edilmiştir, enjektörün içine geri hortum Fluorinert geri çekmek. Parçalama, üst kısmında bulunan atık hortum dahil olmak üzere her bir parçası (tüp delici ve akış hücresi) çıkarın ve su ile iyice durulayın. </li></ol> 6. Temsilcisi Sonuçlar <img src="/files/ftp_upload/1948/1948fig1.jpg" alt="Şekil 1" /> Şekil 1 Siklohekzimidsiz varlığı maya suşu BY4741 (mat his3 Δ 1 leu2 Δ 0 met15 Δ Δ 0 0 ura3) hazırlanan ribozom özü Temsilcisi iz. Ribozomal özü 7% 47 sukroz gradient kullanan fraksiyone ve UV detektörü bağlı bir pompa şırınga aparatı kullanılarak analiz edildi. Polyribosomes ve 80S, 60S ve 40S ribozom içeren Peaks gösterilir.

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

Polyribosome profilden elde edilen bilgiler, hücre translasyonel durumu içine değerli bilgiler sağlayabilir. Buna ek olarak, ribozomal alt birimden kendilerini montaj durumu 2 tespit edilebilir. Örneğin halfmers veya 80S ve profil üzerinde sağa doğru hafif bir omuz daha büyük zirveleri varlığı bağlı 40S katılmadan 60S bekleyen subunit subunit gösterir. Sahne eklenen Siklohekzimidsiz veya diğer uzama inhibitörü yokluğunda deneme uzama 3 değiştirilmiş olup olmadığını gösterir kaçıp oranı, analiz sağlar. Kesirler kendilerini reaktiflerin kesirler, Kuzey ya da Batı blot ribozomal alt popülasyonlar belirli bir mRNA veya protein dernek sonraki belirlenmesi için zengin bir kaynak. Aktif ribozom ile ilişkili mRNA&#39;ların toplam havuzu ilişkili bir mikroarray 4 ya da derin bir sıralama analizi 5 üzerinden tespit edilebilir . Protein dernekleri de uygun reaktif 6 bağlayan bir haç ilave edilerek stabilize olabilir. Memeli hücrelerinde Polyribosome analizi de istikrarlı polysomes almak için gerekli koşulları belirleyen en belirgin zorluklar açısından farklı bir protokol olarak söz değer. Literatürde bildirilen tampon sistemler, 7-10 yaygın olarak değişken böylece lizis tamponu hücre türüne özgü optimizasyonu için bir gereklilik olabilir, ya da tampon sistemi ile çalışmak için istekli dikkat kadar önemli olmadığını eşit makul taze lizatları düşük sıcaklıklarda tutulur. Bildiğimiz kadarıyla memeli polysome oluşumunu etkileyen parametreler sistematik bir değerlendirme rapor edilmemiştir.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

eukaryotic polyribosome profile analysis

Protein sentezi, birçok düzeyde düzenlenir karmaşık bir hücresel süreçtir. Örneğin, küresel çeviri, amino asit, açlık ya da büyüme faktörü çekilmesi gibi hücresel stresler çeşitli başlatma aşamasında veya uzama fazı inhibe olabilir. Alternatif olarak, bireysel mRNA&#39;ların çeviri mRNA lokalizasyonu veya cognate microRNA varlığı ile düzenlenir. Protein sentezi çalışmaları sık sık çeviri düzenleme ya da protein sentezi hatalarına mekanizmalarına ışık tutacak polyribosome analiz kullanmaktadır. Bu testte, mRNA / ribozom kompleksleri ökaryotik hücreler izole edilir. Bir sakaroz yoğunluk gradiyenti tek bir ribozom veya monosome bağlı mRNA&#39;ların polyribosomes olarak bilinen çok sayıda ribozom bağlı mRNA&#39;ların ayırır. Gradyanlar Fraksiyonu farklı ribozomal nüfus ve ilişkili mRNA&#39;ların veya protein ölçümü ve izolasyon sağlar. Tanımlanmış koşullar altında monosomes polyribosomes oranı farklılıklar çeviri başlangıcı ya da uzama / sonlandırma ya hatalarına göstergesi olabilir. Polyribosome kesirler mRNA&#39;ların Sınav bireysel mRNA&#39;ların kohort deneysel koşullar değişiklik tercüme olmadığını ortaya çıkarabilir. Buna ek olarak, ribozom montaj Eðim ayrılmış küçük ve büyük ribozomal altbirim doruklarına analizi ile izlenebilir. Bu video, maya hücreleri, sukroz gradient özü ayırma ve sonuçların yorumlanması ham ribozomal özler hazırlanması için bir yöntem mevcut. Bu prosedür, memeli hücreleri kolayca uyarlanabilir.

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

Bu makale, ökaryotik hücrelerde ribozom tercüme çıkarılması için bir protokol tanımlamaktadır. Bir kez çıkarılan, ribozom, farklı ribozomal popülasyonlar analiz izin vermek için sukroz gradient fraksiyonlandırmasıyla monosomes ve polyribosomes içine ayrılır. Gibi, bu yöntem çeviri düzenleme incelenmesi için altın standarttır.

Ökaryotik Polyribosome Profil Analizi

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Research

JoVE Journal

Biology

52.5K Görüntüleme.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. Bu makale, ökaryotik hücrelerde ribozom tercüme çıkarılması için bir protokol tanımlamaktadır. Bir kez çıkarılan, ribozom, farklı ribozomal popülasyonlar analiz izin vermek için sukroz gradient fraksiyonlandırmasıyla monosomes ve polyribosomes içine ayrılır. Gibi, bu yöntem çeviri düzenleme incelenmesi için altın standarttır.

Video: Eukaryotic Polyribosome Profile Analysis

Immunoblot Analysis

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Immüno Analizi

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the ...

Biyoloji

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

C2C12 Myoblast Hücreler kullanarak Bioenerjetik bilgileri Deney

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Bir Aracı Olarak Korelatif Işık ve Elektron Mikroskobu (CLEM) microinjected Moleküller ve Ökaryotik Alt hücresel Hedefler Visualize&#39;a


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Karbon Fiksasyon Potansiyel bir Kimyasal Tabakalı Antarktika Gölü ve Ölçme Mikrobiyal Ökaryotik Zenginleştirme Kültürlerinin Kurulması

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Zebra balığı Embriyolar Metabolik Profil Analizi

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Metabolik Profil Gerçek Zamanlı Analizi<em&gt; Ex vivo</em&gt; Fare Bağırsak Crypt organoid Kültürleri

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Ökaryotik Hücrelerde gelen ekstrakromozomal dairesel DNA genom Arıtma

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Su Macroinvertebrates kullanma gruba özel rDNA astar genetik Gut içerik analizleri için laboratuvar iletişim kuralı

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Elektron mikroskobu (SEM) tarama morfolojik analiz için kimyasal kurutma kullanarak prokaryotik ve ökaryotik organizmaların hazırlanması

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...