Biz nazik pUTKan bitki ekspresyon vektörü olarak inşa FRET glutamin temini için plazmid ve Dr Uwe Ludewig (Hohenheim Üniversitesi) Fret nazik glikoz ile bize sağladığı için Profesör Kurt Frommer (Bilim Carnegie Enstitüsü, Stanford Üniversitesi) teşekkür ederim. AB BBSRC Hedefli Öncelik Studentships programı tarafından finanse edilmektedir. CK ve KP Hem Biopreparatlar İşleme RCUK Bursu tarafından desteklenmektedir. CK da mali destek için Lonza Biologies teşekkür etmek istemektedir. Sentetik Biyoloji ve Yenilik Merkezi cömertçe EPSRC tarafından desteklenmektedir.

1.. Hücre Hattı Revival ve Bakım <ol><li> 8 mM L-glutamin ve 10 ml / L 100x hipoksantin / timidin takviyesi (tam büyüme ortamı) ile takviye 9 ml CD-CHO ortamı içinde CHO hücreleri canlandırmak. </li><li> 5 dakika boyunca 100 x g&#39;de santrifüj. </li><li> Taze tam büyüme ortamında 10 ml hücreleri yeniden süspanse edin. </li><li> 1 ml&#39;lik bir numune alın ve bir hemasitometre içinde tripan mavi boya eksklüzyon metodunu kullanarak, ışık mikroskopisi ile viyabl hücre konsantrasyonunu belirler. </li><li> 125 ml 3 x 10 5 hücre / ml &#39;lik bir yoğunlukta bir tohum kültürünü başlatmak çalkalama cam deney şişesinin. </li><li> 120 rpm&#39;de dönen bir yörüngesel çalkalama platformu üzerinde, 37 ° C,% 5 CO2 bulunan nemli bir atmosferde bir inkübatörde kültür koruyun. </li><li> 2 x 10 5 hücre / ml &#39;lik bir tohumlama yoğunluğunda tam büyüme ortamı içinde her 3-4 günde bir alt kültür. </li></ol> 2. Plasmid C Hücre Hattı transfeksiyonuBiosensor Gene ontaining <ol><li> Bir büyük ölçekli plazmid saflaştırma kiti kullanılarak plazmid DNA hazırlayın. </li><li> (37 ° C,% 5 CO2), transfeksiyondan önce 12-24 saat hücreler aktif transfeksiyon anda bölünen sağlamak için uygun koşullar altında hücrelerin koruyun. </li><li> Tripan mavi boya eksklüzyon metodunu kullanarak, hücre sayımı, daha sonra 1 x 10 6 hücre / ml &#39;lik bir konsantrasyonda bir sallama şişesi içinde 125 ml hücre kültürü, 20 ml bir çalışma hacmini hazırlayın. </li><li> Kullanılan hücre hattı için uygun bir transfeksiyon kiti kullanın. Transfeksiyon reaktifleri oranı plasmid DNA hücre çizgisi ve vektör üzerindeki bağımlı olacaktır. Bu önceki son transfections yürütülmesi için üretici tarafından belirtilen aralık dahilinde oranların bir dizi test ile optimize önerilir. Ayrıca hücrelerin ancak DNA ihtiva eden en az bir negatif kontrol kültürü korumak. </li><li> Statik modu, 4 gün boyunca inkübe edin ve daha sonra bir transfer Şeki125 rpm&#39;de dönen ng platformu. </li><li> Bir öldürme eğrisi tarafından tespit edildiği üzere, uygun bir konsantrasyona kadar plazmid seçimi için uygun antibiyotik ekleyin. Bu çalışmada, zeosin transfekte edilmiş hücrelerin seçilmesi için 400 ug / ml &#39;lik bir nihai konsantrasyona kadar ilave edildi. </li><li> Kontrol çukurdaki hücre ölmüş kadar, antibiyotik her zaman bir ekleyerek, hücre hattı için uygun bir zaman aralığında ortam kullanın. </li><li> Kültürleri sallayarak ilerleme için en Konfluent kuyuları kullanın. </li><li> Dondurma karışımı 1 ml 10 7 canlı hücreleri içeren şişeler içinde kriyojenik dondurma hücreleri tarafından hücre bankası kurulması. Bu çalışmada, bu% 92.5 büyütme ortamı ve% 7.5 dimetil sülfoksit oluşur. </li></ol> 3. Toplu ve Fed-batch Cell Büyüme Eğrisi <ol><li> 50 ml çalışma hacmine sahip 250 ml&#39;lik çalkalama cam deney şişesinin içinde transfekte edilmiş CHO hücrelerinin hücre kültürlerinin, üçlü kurmak için yukarıdaki hücre bakım talimatları takip edin. </li><li> Cultu koruyunnemli bir hücre 125 rpm&#39;de dönen bir yörüngesel çalkalama platformu üzerinde,% 5 CO2 ile 37 ° C&#39;de kuluçka, ve 24 saatlik aralıklarla her bir büyüme kültüründen 4.1 ml örnekleri kaldırmak de res. </li><li> Tripan mavi boya eksklüzyon yöntemi ile, viyabl hücre yoğunluğu ve hücre yaşayabilirliğini belirlemek için numune 100 ul kullanın. </li><li> Viyabl hücre konsantrasyonu sıfıra indirgenene kadar tekrar edin. </li><li> Beslemeli-kesikli hücre kültürleri, sırasıyla 36 mM ve 4 mM başlangıç ​​değerlerine şekilde bunların konsantrasyonlarını geri 6. günde glukoz veya glutamin, uygun miktarda takviye için tekrarlayın. Bu, saf suyun aynı hacimde (bu durumda 12 mi) ile beslenmeleri için olan ek kontrol kültürleri de korunur önerilir. </li></ol> 4. Oranı Ölçümleri FRET <ol><li> 4 ° C&#39;de 5 dakika boyunca 100 x g yukarıdaki aşama 3.2 ve santrifüj altında uzaklaştırıldı günlük numunelerin 2 ml alın </li><li> Resuspbuz soğuğu fosfat tamponlu tuzlu su (PBS) içinde bir 2 ml hücre pelletini sona erdi. 6 oyuklu bir plakaya aktarılır ve bu da bir hücre içeren herhangi bir boş örnek ekleyin. </li><li> Derhal 465/435 nm (mavi), 520/510 nm (sarı) 430/435 nm ve emisyon dalga boylarındaki bir uyarma dalga boyunda ve sarı, mavi floresan seviyeleri ölçülür. </li><li> FRET oranları (algılanan mavi floresan yerinde tespit sarı floresan oranı) hesaplayın. </li></ol> 5. Metabolit Tahliller <ol><li> 4 ° C&#39;de 5 dakika boyunca 100 x g yukarıdaki aşama 3.2 ve santrifüj altında uzaklaştırıldı günlük numunelerin 2 ml alın Süpernatant kaldırmak. </li><li> 5 dakika boyunca 100 x g hızında tekrar 3 ml buz gibi soğuk PBS ve santrifüj hücre pelletini. </li><li> Süpernatantı ve yukarıdaki gibi 3 ml buz gibi soğuk PBS ile hücre pelletini. 15 saniyelik bir darbe de 3 dakika, her 15 saniye için kapalı örnek 5x sonikasyon. Bu noktada, hücre ilaveİsterseniz cts donmuş olabilir. </li><li> (Malzemelerin tabloya bakınız) hücre içi glikoz konsantrasyonunu belirlemek için, hücre ekstrelerinin örnekler üzerinde üreticinin talimatlarına uygun olarak, uygun bir glükoz tahlil kiti kullanın. Not: bir standart eğri oluşturmak için uygun standartları kullanın. Bu çalışmada, standartların konsantrasyonu 0-100 mM arasında değişmektedir. </li><li> Standartları için okumalar kullanarak, standart eğri oluşturmak ve (bu durumda: y = 1310.51x - y absorbans okuma ve x, glikoz konsantrasyonu olan 723,43) en uygun doğrusal denklemi bulabilirsiniz. </li><li> , Standart eğri kullanılarak ve dikkate alınarak seyreltme glikoz konsantrasyonları hesaplayın. Hücre içi glikoz konsantrasyonu daha sonra, bu denklem kullanılarak hesaplanabilir: <img alt="" fo:content-width="4.5in" src="/files/ftp_upload/51200/book_bg.png" width="500" /> CHO hücreleri için, tek bir hücre hacmi, 12 uM, 12 &#39;lik bir hücre çapı kullanılarak hesaplanmıştır </sup&gt;. </li><li> (Malzemelerin tabloya bakınız) hücre içi glutamin konsantrasyonunu belirlemek için, hücre ekstrelerinin örnekler üzerinde üreticinin talimatlarına uygun olarak, uygun bir glutamin deney kiti kullanın. Not: bir standart eğri oluşturmak için uygun standartları kullanın. Bu çalışmada, standartların konsantrasyonu 0-2 mm arasında değişmektedir. </li><li> Yukarıda 5.5 &#39;de olduğu gibi, (y absorbans okuma ve x, y = 203.1x + 17.1 glikoz konsantrasyonu bu durumda) kalibrasyon eğrisi oluşturmak </li><li> Standart eğri kullanılarak, dikkate alınarak seyreltme, numunelerin glutamin konsantrasyonunu hesaplamak. Hücre içi glutamin konsantrasyonu, yukarıdaki denklem 1 kullanılarak hesaplanabilir. </li></ol>

<ol>
	<li>Moser, I., Jobst, G., Urban, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensor+arrays+for+simultaneous+measurement+of+glucose,+lactate,+glutamate,+and+glutamine.">Biosensor arrays for simultaneous measurement of glucose, lactate, glutamate, and glutamine.</a> Biosens. Bioelectron. 17 (4), 297-302 (2002).</li><li>Billingsley, K., 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=Fluorescent+nano-optodes+for+glucose+detection.">Fluorescent nano-optodes for glucose detection.</a> Anal. Chem. 82 (9), 3707-3713 (2010).</li><li>Dattelbaum, J. D., Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Determination+of+Glutamine+Using+a+Genetically+Engineered+Protein.">Optical Determination of Glutamine Using a Genetically Engineered Protein.</a> Anal. Biochem. 291 (1), 88-95 (2001).</li><li>Kfouri, M., 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=Toward+a+miniaturized+wireless+fluorescence-based+diagnostic+imaging+system.">Toward a miniaturized wireless fluorescence-based diagnostic imaging system.</a> IEEE J. Sel. Top. Quantum Electron. 14 (1), 226-234 (2008).</li><li>Dittrich, P. S., Manz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+detection+in+microfluidic+channels&#8212;the+Holy+Grail+in+muTAS.">Single-molecule fluorescence detection in microfluidic channels&#8212;the Holy Grail in muTAS.</a> Analytical and Bioanalytical Chemistry. 382 (8), 1771-1782 (2005).</li><li>Hou, B. H., Codamo, J., Pilbrough, W., Hughes, B., Gray, P. P., Munro, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sensors+for+monitoring+dynamic+changes+of+intracellular+metabolite+levels+in+mammalian+cells.">Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells.</a> Nat. Protoc. 6 (7), 1818-1833 (2011).</li><li>Bermejo, C., Haerizadeh, F., Takanaga, H., Chermak, D., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+cytosolic+glucose+and+ATP+levels+in+yeast+using+optical+sensors.">Dynamic analysis of cytosolic glucose and ATP levels in yeast using optical sensors.</a> Biochem. J. 432, 399-406 (2010).</li><li>Yang, H. Y., Bogner, M., Stierhof, Y. D., Ludewig, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H(+)-independent+glutamine+transport+in+plant+root+tips.">H(+)-independent glutamine transport in plant root tips.</a> Plos One. 5, (2010).</li><li>Fehr, M., Takanaga, H., Ehrhardt, D. W., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+high-cavacity+bidirectional+glucose+transport+across+the+endoplasmic+reticulum+membrane+by+genetically+encoded+fluorescence+resonance+energy+transfer+nanosensors.">Evidence for high-cavacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors.</a> Mol. Cell. Biol. 25 (24), 11102-11112 (2005).</li><li>Ai, H. W., Hazelwood, K. L., Davidson, M. W., Campbell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET+pairs+for+ratiometric+imaging+of+dual+biosensors.">Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors.</a> Nat. Methods. 5 (5), 401-403 (2008).</li><li>Ouyang, M. X., Sun, J., Chien, S., Wang, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+hierarchical+relationship+of+Src+and+Rac+at+subcellular+locations+with+FRET+biosensors.">Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors.</a> Proc. Natl. Acad. Sci. U.S.A. 105 (38), 14353-14358 (2008).</li><li>Han, Y., 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=Cultivation+of+recombinant+Chinese+hamster+ovary+cells+grown+as+suspended+aggregates+in+stirred+vessels.">Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels.</a> J. Biosci. Bioeng. 102 (5), 430-435 (2006).</li><li>Deuschle, K., Okumoto, S., Fehr, M., Looger, L. L., Kozhukh, L., Frommer, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+optimization+of+a+family+of+genetically+encoded+metabolite+sensors+by+semirational+protein+engineering.">Construction and optimization of a family of genetically encoded metabolite sensors by semirational protein engineering.</a> Protein Sci. 14 (9), 2304-2314 (2005).</li><li>Wong, D. C. F., Wong, N. S. C., Goh, J. S. Y., May, L. M., Yap, M. G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+N-Glycosylation+gene+expression+in+CHO+cell+Fed-batch+cultures.">Profiling of N-Glycosylation gene expression in CHO cell Fed-batch cultures.</a> Biotechnol. Bioeng. 107 (2), 516-528 (2010).</li><li>Behjousiar, A., Kontoravdi, C., Polizzi, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Situ+Monitoring+of+Intracellular+Glucose+and+Glutamine+in+CHO+Cell+Culture.">In Situ Monitoring of Intracellular Glucose and Glutamine in CHO Cell Culture.</a> PLoS One. 7, (2012).</li><li>Dwyer, M. A., Hellinga, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periplasmic+binding+proteins:+a+versatile+superfamily+for+protein+engineering.">Periplasmic binding proteins: a versatile superfamily for protein engineering.</a> Curr. Opin. Struct. Biol. 14 (4), 495-504 (2004).</li></ol>

Yazarlar ifşa hiçbir şey yok.

FIBS yöntem söndürülmesi ve ekstraksiyon kaynaklanan engelleyerek Bu durumda, büyüme sınırlayıcı besin in, in vivo ve in önemli moleküllerin in situ olarak nicelendirilmesi sağlar. Bulgular FIBS sinyal ve glutamin için glikoz ve 0,3-2 mm 1-5 mM aralığı içinde, hücre içi konsantrasyonları arasında iyi bir ilişki olduğunu ortaya koymaktadır. Toplu CHO hücre kültürü, bu konsantrasyonlar, üstel sabit ve erken aşamalarında düşüş rastlanmaktadır. Üstel ve durağ...

Metaboliti izleme süreç geliştirme, medya ve yem tasarım ve metabolik mühendislik dahil bioprocessing çeşitli uygulamalar vardır. Çeşitli yöntemler enzimatik 1, kimyasal 2, 3 ya da bağlama deneylerinde kullanımıyla konsantrasyonu ölçümleri için kullanılabilir. İlginç bir seçenek, floresan kullanılmasıdır, protein bazlı anahtar metabolitlerin hücre içi konsantrasyonunun muhabir olarak in vivo biyolojik sensörler. Küçültme aslında sinyal-gürültü oranı, 4,5 artırır ve protein bazlı sensörler eksojen genetik olarak reaktif metabolit analizi için gerekli olduğu anlamını kodlanabilir gibi ultra-düşük hacim deneylerinde, floresan bir araçtır. Förster rezonans enerji transferi (FRET) biyosensörler bir ligand bağlama alanı ile bağlanmış iki floresan protein oluşur. FRET bir alıcı, flüoresan molekül locat bir fotoğraf eksitasyonlu bir vericiden bir radyatif olmayan enerji transferi<html> yakın (&lt;100) ed. Ligand bağlayıcı bir bölünme ya da sırayla, emisyon spektrumunda bir değişiklik ile ölçüldüğü FRET veriminde bir değişime yol açan, florofor yakın bir değişim olmaktadır, sensörün bir konformasyonel değişikliği neden olur. FRET entegre biyolojik sensörleri (FIBS) yapıcı hücre hattı içinde üretilir ve spektral özellikleri, hücre içinde metabolitin konsantrasyonuna göre değişir. FIBS dinamik modellerin 6 ölçümler için idealdir hızlı tepki süreleri var. Önceki uygulamaların 7-9 tekli ve çoklu 10 metabolitleri izleme ve uzaysal dağılımı 11 veri sağlayan içerir. Ligand bağlama enerjisi transferi ve bir ligand bağlanma yakın temas eder (Şekil 1) içine, iki florofor getirir Apo-Min, düşürücü florofor yakınlığını bozan Apo-Max,: FIBS iki konfigürasyonlarda oluşturulabilir. Bu çalışmada _content &quot;&gt;, bir protokol kurucu bir metaboliti, glukoz ya da glutamin için bir ifade fibs (CHO) hücre çizgileri, Çin hamsteri yumurtalık oluşturmak için sunulmuştur. bir yöntem, in vivo olarak, sensörün kalibrasyonu için kurulur gelecek Quantitative etkinleştirme Şekil 2&#39;de gösterildiği gibi. Buna göre hücre içi metabolit konsantrasyonunun ölçümleri, glukoz veya glutamin hücre içi konsantrasyonu, iki besin işlemdeki yüksek konsantrasyonlarda ilave edildi için, beslemeli-kesikli CHO hücre kültürlerinde tespit edilebilir. sonuçlar Bu invaziv olmayan, düşük maliyetli olduğundan bağımsız enzimatik deneyi ile teyit edildiği gibi FIBS ve daha önceden oluşturulan kalibrasyon eğrisinden floresan sinyali kullanarak, hücre içi konsantrasyonunun doğru tahmini mümkün olduğunu göstermektedir. bu yöntem mevcut analitik teknolojiler üzerinde önemli avantajlar sunar ve hızlı mon olabilir FIBS bir gerçek-zamanlı sinyal vererekkültür boyunca itored.

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			<td height="22" style="height:22px;width:239px;">CHO-S Cells</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;">11619-012</td>
			<td style="width:459px;">Cell line will vary depending on the goals of the study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">pCDNA4/TO vector&#160;</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">TransIT-PRO Transfection Reagent</td>
			<td style="width:120px;">Mirus Bio</td>
			<td style="width:120px;">MIR 5700</td>
			<td style="width:459px;">Transfections can be accomplished using any method suitable for the cell line under study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Zeocin</td>
			<td style="width:120px;">Invivogen</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">CD-CHO medium</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">10743-011</td>
			<td style="width:459px;">Cell growth medium is dependent upon the cells under study</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">100X HT Supplement</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">11067-030</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">L-Glutamine 200 mM (100X), Liquid</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">25030032</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">InfinitePRO 200 plate reader</td>
			<td style="width:120px;">Tecan</td>
			<td style="width:120px;">FLx800TBI</td>
			<td style="width:459px;">Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted</td>
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			<td height="190" rowspan="6" style="height:190px;width:239px;">Filters for plate reader</td>
			<td rowspan="6" style="width:120px;">Tecan</td>
			<td align="right" style="width:120px;">30000463</td>
			<td rowspan="6" style="width:459px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(520NM BW 10NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(430NM BW 35NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022787</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">(465NM BW 35NM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Maxiprep Plasmid Purification Kit</td>
			<td style="width:120px;">Qiagen</td>
			<td style="width:120px;">12163</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Amplex Red glucose/glucose oxidase assay kit</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:120px;">A22189</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">EnzyChrom&#160; Glutamine Assay Kit</td>
			<td style="width:120px;">BioAssay systems</td>
			<td style="width:120px;">EOAC-100</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Improved Neubauer haemocytometer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:120px;">MNK-420-010N</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Incubator&#160;</td>
			<td style="width:120px;">Nuaire</td>
			<td style="width:120px;">NU-5510E</td>
			<td style="width:459px;"></td>
		</tr>
	</tbody>
</table>

fibs-enabled noninvasive metabolic profiling

Hesaplamalı biyoloji çağında, yeni yüksek kapasiteli deneysel sistemler onlar tahmin amacıyla valide böylece modelleri doldurmak ve rafine etmek için gereklidir. İdeal tür sistemler zaman ders veri elde edilecek zaman örnekleme ve yıkıcı analizlerini engeller düşük hacimli olacaktır. Gereken şey, gerçek-zamanlı ve noninvaziv gerekli bilgileri rapor edebilecekleri in situ izleme aracıdır. İlginç bir seçenek, floresan kullanılmasıdır, protein-bazlı hücre içi konsantrasyonlarının muhabir olarak in vivo biyolojik sensörler. Metabolit nicellendirmesinde bulmuştur in vivo biyosensör bir özel sınıfı, bir ligand bağlama alanı ile bağlanmış iki floresan protein arasında Förster rezonans enerji transferi (FRET) dayanmaktadır. Entegre biyolojik sensörler (FIBS) Fret yapıcı hücre hattı içinde üretilen, onlar hızlı yanıt süreleri ve spektral cha varhücre içindeki metabolitin konsantrasyonuna göre değişiklik tikleri. Bu yazıda, hücre içi metabolit konsantrasyonunun gelecek ölçümü sağlamak amacıyla yapısal olarak glikoz ve glutamin için bir ifade fibs (CHO) hücre çizgileri, Çin hamsteri yumurtalık yapımında ve toplu hücre kültüründe in vivo olarak fibs kalibre edilmesi için bir yöntem tarif edilmektedir. Beslemeli-kesikli CHO hücre kültürlerinden elde edilen veriler, FIBS hücre içi konsantrasyonunda elde edilen değişikliği tespit etmek için, her durumda mümkün olduğunu göstermektedir. Bağımsız bir enzimatik deneyi ile teyit edildiği gibi FIBS ve daha önceden oluşturulan kalibrasyon eğrisinden floresan sinyalini kullanarak, hücre içi konsantrasyonu tam olarak tespit edilmiştir.

Metodolojinin bir bakış, Şekil 2&#39;de sunulmuştur. Burada sunulan çalışma olarak, CHO hücreleri FIBS vektör ve stabil hücre hatları ile transfekte edilmiş 400 ug / ml zeosin bir antibiyotik basınçta seçildi. Yapısal olarak glikoz ve glutamin sensörler sentezleyen iki ayrı kararlı hücre soyları bu nedenle, yaratılmıştır. Şekil 1, bu çalışmada kullanılan iki biyosensör konfigürasyonlarını göstermektedir. Glikoz sensörü Apo-Max prensibine dayanan ve m...

Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

FIBS-enabled Noninvasive Metabolic Profiling

In situ metabolik profilleme için Förster Rezonans Enerji Transferi entegre biyolojik sensörler (FIBS) kalibre nasıl bir açıklama sunulmuştur. FIBS invaziv olmayan metabolik modelleri ve biyoproses koşullarının yüksek miktarda madde taraması gelişiminde yardım metabolitlerin hücre içi seviyelerini ölçmek için kullanılabilir.

Noninvaziv metabolik profil FIBS etkin

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

fibs-enabled-noninvasive-metabolic-profiling

Research

JoVE Journal

Bioengineering

9.6K Görüntüleme.  Imperial College London.  In situ metabolik profilleme için Förster Rezonans Enerji Transferi entegre biyolojik sensörler (FIBS) kalibre nasıl bir açıklama sunulmuştur. FIBS invaziv olmayan metabolik modelleri ve biyoproses koşullarının yüksek miktarda madde taraması gelişiminde yardım metabolitlerin hücre içi seviyelerini ölçmek için kullanılabilir. 

Video: FIBS-enabled Noninvasive Metabolic Profiling

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

Yüksek Verimli MicroRNA Profil: Optimize edilmiş, Multiplex QRT-PCR Fluidigm Dinamik ArrayTM IFCS Nanoliter Ölçeğinde

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Biyomühendislik

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

Yerinde Basınç Yükleme ve Kemik-periodontal ligament-diş Lifli Joint Korelatif noninvaziv Görüntüleme

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Sayımı ve Ayarlanabilir Dirençli Darbe Algılama Yöntemi Ekstrasellüler Veziküller boyutu-profil

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Micropost Diziler Kolay ve Doğru Mekanik-profil

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Sialik asit Nkullanarak metabolik Glycoengineering-asil-Mannosamines modifiye

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

Metabolik destek çıkardım, yaşam beyin dokuları manyetik rezonans mikroskobu Alım sırasında

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: Hücresel enerji metabolizması araştırmak için bir güçlü, invaziv olmayan bir yaklaşım

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensors için noninvaziv tanı koyma proteaz aktivitesi Vivo tespit

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Küresel hücresel matriks metalloproteinaz ve 3D Hydrogels metabolik etkinliğini ölçme

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Bir İnsan 3D Ekstrasellüler Matrix-Adiposit Kültür Modeli Matrix-Hücre Metabolik Crosstalk Çalışma için

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...