Biz C. Caenorhabditis Genetik Merkezi&#39;nden kabul elegans suşları. Finansman desteği Terry Fox Araştırma Enstitüsü tarafından sağlandı. Biz de Rolande ve Marcel Gosselin Lisans Bursu ve McGill Entegre Kanser Araştırma Eğitim Programı kanser araştırma FRN53888 içinde CIHR / FRSQ eğitim hibe EP verilen destek için minnettarım.

Reaktifler 1. Hazırlık <ol><li> C. medyanın hazırlanması (Bu durumda, vahşi tip hayvan ve FLCN-1 (ok975) mutant hayvanlarda) elegans büyümesi. <ol><li> Tris HCI ve 5.5 g Tris baz, 2.4 g, Bactopetone 31 g, NaCl 20 g ve kolesterol 0.08 g &#39;ını ihtiva Youngren tek Bacto-pepton (MYOB), kuru bir karışım Modifiye hazırlayın. Sallayarak iyice karıştırın. NOT: Bu karışım MYOB ortamı 10 L hazırlamak için yeterlidir. Not: normal büyüme ortamı (NGM) plakaları MYOB plakalar 27 yerine kullanılabilir. Bununla birlikte, plakaların aynı tip suşlar arasında bağımsız tekrarlar arasında geçerli bir karşılaştırması için kullanılmalıdır. Bu protokol, biz sadece MYOB plakaları kullanılır. </li><li> MYOB kuru karışımın 6 g, agar 17 g eklenerek MYOB ortamı 1 L hazırlayın ve 122 ° C &#39;de 45 dakika süre ile H2O ve otoklav ile 1 L&#39;ye tamamlanır. Plakaları dökün ve 2 gün boyunca oda sıcaklığında kurumaya bırakın. </li><li> Steril teknik kullanılarak, kültürlerdeki 100 ml&#39;sini aşılamakE. LB sıvı ortamında yeniden E. coli OP50 bakteri ve 37 ° C &#39;de O / N büyür. </li><li> E. Tohum MYOB plakaları E. coli OP50 bakterileri. 48 st için oda sıcaklığında büyümesi izin verin. </li></ol></li><li> RNAi kullanılarak gen baskılanması Hazırlık <ol><li> MYOB RNAi besleme tabak hazırlayın. Adımlar 1.1.1 ve 1.1.2 gibi devam edin. Otoklav sonra, ortam yaklaşık 55 ° C&#39;ye kadar soğutulur ve eklemek için izin 1 M izopropil β-D-1-tiogalaktopiranosid (IPTG) ve 100 mM ampisilin, 1 ml 1 mi. </li><li> Streak E. E. coli HT115 kontrol EV veya agar plakaları ampisilin üzerinde FLCN-1 spesifik RNAi (50 ug / ml) / tetrasiklin (15 ug / ml) ya da ifade eden bir plazmid ile transforme edilmiş bakteriler. 37 ° C&#39;de, kültür O / N büyütün. </li><li> Koloniler seçin ve E. aşılamak E. coli HT115 kontrol EV ya da belirli bir RNAi 1 FLCN ve ya 37 ve # LB / ampisilin (50 ug / ml), orta ve O / N kültürünün yetiştirilmesi eksprese eden bir plazmid ile transforme edilen bakteri176 ° C. </li><li> E. ile Tohum plakalar E. coli HT115 EV bakteri veya HT115 FLCN-1 RNAi bakteri. NOT: Kullanımdan önce oda sıcaklığında plakaları 48 saat tutun. Besleme RNAi demonte etkili ise, DS-RNA 28 teslim diğer yöntemleri kullanabilirsiniz. </li></ol></li><li> Parakuat (PQ) ile oksidatif stres oluşturma: <ol><li> PO 4 KH 2, 3 g karıştırılarak M9 tampon hazırlanması, Na 2 6 g HPO 4, 5 g NaCI ve MgSO 4 0.25 g · 7 H2O Damıtılmış su ile 1 L&#39;ye getirin. Oda sıcaklığında 122 ° C ve mağaza şişe 45 dakika süreyle otoklavlanmaktadır çözeltisi. </li><li> Analiz gününde, M9 / PQ-içeren solüsyon hazırlanır. (M9 4 ml tampon içinde eritildi PQ 0,1028 g oyuk başına M9 / PQ 40 ul ile bütün bir 96 gözlü plakalar doldurur) Bu protokol için, 100 mM PQ kullanın. DİKKAT: PQ tehlikeli ve son derece toksik bir bileşiktir. Uygun kurallarına göre işlemek. NOT: İlk yeni s çalışanTren veya yeni bir durum, bu 7-10 saat içinde hayvanları öldürmek için kullanılacak en iyi konsantrasyonunu belirlemek için PQ artan konsantrasyonları hayatta kalma tahlil için tavsiye edilir. </li></ol></li></ol> Yaş senkronize L4 Nüfus 2. Hazırlık <ol><li> E. ile seribaşı taze MYOB plaka Transferi 20 gebe yetişkin çift cinsiyetli E. coli OP50 bakterileri. Tahlil sırasında gerekli solucanlar sayısına dayalı birkaç tabak hazırlayın. </li><li> Onları 6 saat sonra anneleri kaldırmak platin tel solucan kıracağı kullanarak yumurtalarını bırakmak için izin verin. Bıraktıkları yumurta sayısını değerlendirmek için mikroskop altında plakaları kontrol edin. </li><li> Yumurtaların açılması ve 48 saat boyunca 20 ° C&#39;de büyümeye izin verin. Bu zamanda, hayvanlar, bir geç L4 / genç yetişkin aşamasındadır. Aynı sahne hayvanları seçin ve PQ çözüm içeren kuyulara aktarın. NOT: 48 saat inkübasyon, sadece yabani tipte hayvanlara benzer şekilde büyümeye mutantlar için geçerli olduğundan, bu gelişim takip etmek önemlidirYeni test mutantların oranı, vahşi tip gelişme hızına aksi halde, başka bir zaman çizgisi kullanılmalıdır uygun sağlamak. </li><li> Alternatif olarak, senkronizasyon tekniklerini kullanın. Hipoklorit çözeltisi kullanılarak L1 larvalarının solucanların senkronize popülasyonunu üretmek (25 ml su,% 5.25 sodyum hipoklorit 125 mi, 4 M NaOH, 50 ml, ışıktan izole etmek için alüminyum folyo ile bir şişe sarın). Yoğun bir ağartıcı tahlil sırasında hayvan davranışını etkiler olabilir Bununla birlikte, bu deney için, bu, tavsiye edilmez. DİKKAT: kurallarına göre hipoklorit solüsyonu tutun. Not: aşağı ilgilenilen geni düzenleyen özel RNAi bakteri ile ekim yapılmış RNAi plakalara annelerin transferi hariç olmak üzere, bölüm 2&#39;de tarif edildiği gibi çift cinsiyetli eşitlemeye RNAi kullanırken. RNAi ile demonte zayıf olduğunda, birden nesiller için beslenme tavsiye edilir. </li></ol> 3. Oksidatif Stres Direnci Deneyi &lt;Sahne/ P&gt; <ol><li> Pipet 96 oyuklu bir plakanın her bir oyuğuna (taze elde edilmiş) 100 mM PQ çözeltisi 40 ul. Her durumda (tedavi, mutant, vs.) replike olarak en az 12 kuyu kullanın. Bir platin tel solucan maşası kullanarak, her iyi başlangıç ​​ve bitiş zamanlarını gösteren 5-8 L4 larvaları hayvanları transfer. </li><li> Başka bir koşul başlatırken, başlangıç ​​ve bitiş zamanlarını not edin. Başlangıç ​​zamanından itibaren bir saat sonra ölü hayvanları solucanlar aktarılması ve puanlama arasındaki inkübasyon süresi içinde 20 ° C&#39;de plaka koyun. </li><li> Diseksiyon mikroskobu kullanarak, hayatta kalma her saat saymak. Yavaşça saymak başlamadan önce plakayı sallayın. Ölü hayvanlar gibi, hatta yüksek yoğunluklu ışık lekelenme sonra, hareket etmeyen solucanlar belirtiniz. Ayrıca canlı hayvanların sayısını göstermektedir. NOT: Yüksek büyütmede solucan şekli, kuyrukları ve baş hareketi baktığımızda, diri ölü solucanlar belirlemeye yardımcı olur. </li><li> En solucanlar ölene kadar bu adımı her saat tekrarlayın. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Every Time Point Survival Yüzde Belirlenmesi <ol><li> Iyi her transfer hayvanların toplam sayısı ekleyerek durum başına toplam hayvan sayısını hesaplayın. Hasarlı veya transfer etmesi halinde öldürülen hayvan geçiyoruz. </li><li> Her zaman noktası için, (12 kuyuda ölü hayvanların toplamı) durumuna başına ölü hayvanların toplam sayısını hesaplamak. Her zaman noktası için, ve hayatta kalma (100 eksi yüzde ölümü) yüzdesi (ölü hayvanlar 100 ile hayvanların toplam sayısına bölünmesiyle ve çarpılır sayısı) ölüm yüzdesini hesaplar. </li><li> Bu deney en az 3 bağımsız kez tekrarlayın. </li></ol>

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S., Curtis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AMP-activated+protein+kinase+AAK-2+links+energy+levels+and+insulin-like+signals+to+lifespan+in+C.+elegans.">The AMP-activated protein kinase AAK-2 links energy levels and insulin-like signals to lifespan in C. elegans.</a> Genes Dev. 18, 3004-3009 (2004).</li><li>Lee, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+AMP-activated+protein+kinase+AAK-2+is+phosphorylated+by+LKB1+and+is+required+for+resistance+to+oxidative+stress+and+for+normal+motility+and+foraging+behavior.">The Caenorhabditis elegans AMP-activated protein kinase AAK-2 is phosphorylated by LKB1 and is required for resistance to oxidative stress and for normal motility and foraging behavior.</a> J Biol Chem. 283, 14988-14993 (2008).</li><li>Honda, Y., Honda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+daf-2+gene+network+for+longevity+regulates+oxidative+stress+resistance+and+Mn-superoxide+dismutase+gene+expression+in+Caenorhabditis+elegans.">The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans.</a> FASEB J. 13, 1385-1393 (1999).</li><li>Restif, C., Metaxas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+swimming+motions+of+C.+elegans+worms+with+applications+in+aging+studies.">Tracking the swimming motions of C. elegans worms with applications in aging studies.</a> Med Image Comput Comput Assist Interv. 11, 35-42 (2008).</li><li>Buckingham, S. D., Sattelle, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+automated+measurement+of+nematode+swimming+(thrashing)+without+morphometry.">Fast, automated measurement of nematode swimming (thrashing) without morphometry.</a> BMC Neurosci. 10, 84 (2009).</li></ol>

The authors declare that they have no competing financial interests.

C. elegans kolaylıkla kültürlenebilir yana in vivo genetik olarak oksidatif stres direnci çalışma için çekici bir model organizma ve hızlı bir şekilde genetik olarak özdeş yavruların çok sayıda yol açar. Oksidatif stres direncini ölçmek için birden çok yöntem, daha önce tarif edilmiştir ve böyle PQ, rotenone, H2O 2 ve juglon 25,26,29-32 gibi çeşitli ROS kaynakları ile kültür levhaları takviyesi dayanmaktadır. Burada 100 mM PQ kullanılarak 96 yuval?...

A reference was corrected in the publication of <a href="http://www.jove.com/video/52746">Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates</a>. There was an error with reference 34 in the Citations section. It has been updated from:
Allen, E., Walters, I.B., Hanahan, D. Brivanib a dual FGF/VEGF inhibitor,is active both first and second line againstmouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance toVEGF inhibition. Clin Cancer Res. 17, (16) 5299-310 (2011).
to:
Allen, A. T., Maher, K. N., Wani, K. A., Betts, K. E., &#38; Chase, D. L. Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans. Genetics. 188, 579-590 (2011).

Erratum: Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Ökaryotlarda, mitokondri elektron taşıma zincirinde yer alan oksidatif fosforilasyon ATP şeklinde enerji üretimi temel sürücüdür. Reaktif oksijen türleri (ROS) Bu işlem doğal bir yan ürünü bulunmaktadır. Sinyal molekülleri olarak önemli bir rolü olmasına rağmen, aşırı ROS DNA hasarı protein karbonilasyonu ve lipid oksidasyonuna neden olabilir. ROS üretimi ve detoksifikasyon arasında bir dengesizlik enerji tükenmesi, hücresel hasara yol açan oksidatif strese neden olur ve hücre ölümünü 1,2 tetikler. Oksidatif stres, yaşlanma ve kanser, diyabet, kalp-damar ve nörodejeneratif hastalıklar 3-9 dahil olmak üzere birçok yaşamı tehdit eden hastalıkların gelişimine katkıda bulunur. Hücreler uygun ROS düzeylerini korumak için ve oksidatif hasara karşı 1,2 bileşenlerinin korumak için enzimatik ve non-enzimatik savunma stratejilerini geliştirmişlerdir. Süperoksit dismutaz (SOD) enzim conve ilk hareketDaha sonra, katalaz veya peroksidaz enzimleri tarafından suya dönüştürülür H2O 2, RT süperoksit. Sigara enzimatik savunma stratejilerinin daha çok temel hücresel bileşenleri korumak makromoleküllerle, karşılaştırıldığında ROS ile daha hızlı reaksiyona molekülleri içerir. ROS enzimler detoks koruyucu rolüne rağmen, bazı ROS molekülleri antioksidan savunma mekanizmalarını kaçış ve oksidatif hasara yol açar. Algılama, onarım ve hasarlı hücre bileşenlerinin bozulması oksidatif strese 1,2 sırasında gerekli savunma stratejileri vardır. Stres direnci ve özellikle oksidatif stres yer alan sinyal yolları çok evrimsel 10,11 muhafaza edilir. Organizma koşullar sadece kısmen çoğaltılmış hücre kültürü deneyleri, 12,13 büyük önem taşımaktadır model organizmaların oksidatif stresin çalışmanın aksine. C elegans cul kolay ve ucuz olabilir serbest yaşayan nematod olanAgar medya üzerinde bir bakteri çim tured. Küçük boyutta normal olarak (yaklaşık 1 mm uzunluğunda) ve genetik manipülasyonlar kolaylaştıran kendiliğinden gübre hermafrodit olarak büyür. Bu hızlı bir yaşam döngüsü ve yüksek üreme kapasitesine, nesilden başına yaklaşık 300 yavru üreten o büyük ölçekli genetik ekranlar 14 gerçekleştirmek için güçlü bir araç yapma vardır. C. C. elegans genomunun tam dizilenmiş ve genlerin% 40-50, insan hastalığı ile ilişkili genlerin 15-18 homologları olduğu tahmin edilmektedir. RNAi kullanılarak ilgi genlerinin yok etme, hızlı ve C kolay elegans. Gene aşağı düzenleme E. hayvan besleme ile elde edilebilir İlgi 19 mRNA hedef çift iplikçikli RNA eksprese eden bir plazmide E. coli bakterileri. Bu nedenle, büyük ölçekli RNAi ekranlarını kullanarak gen fonksiyonu belirlenmesi kanseri 20,21 dahil olmak üzere insan hastalıklarının anlaşılması üzerinde büyük etkisi vardır. O çalışmalarıC. xidative stres direnci elegans oksidatif stres 13,22 direnç muhafaza mekanizmaları tanımlanmasına yol açmıştır. Belirlenen bazı yollar hipoksi, ısı ve ozmotik stres gibi diğer stresler yanı gibi uzun ömürlü ve direnci modüle ortak yollar vardır. Bu yollar, insülin sinyalizasyon, TOR sinyalizasyon ve Otofaji sayılabilir. Diğer önemli yollar gibi, ya da hasar onarım gibi ısı şoku proteinleri ve şaperon 11,13,22 olarak süperoksit dismutaz enzim ve katalaz enzimleri olarak ROS detoksifikasyon içerir. Bu protokol C. oksidatif strese karşı direnci nasıl belirleneceği açıklanır sıvı içinde elegans. Daha önce C&#39;de kaybı ortaya FLCN-1 (ok975) oksidatif strese karşı artmış bir direnç göstermiştir beri protokol göstermek için FLCN-1 (ok975) ve vahşi tip hayvanlara kullanılan elegans 23. Ayrıca, bu daha iyi direnç gösterme bağlı olduğunu göstermiştirAMPK ve Otofaji hücresel biyoenerjitiklerinin artırır ve gerilme direnci 23 teşvik eden bir sinyal ekseninde. PQ reaktif oksijen türleri 24 üretmek için elektron taşıma zinciri engelleyen bir oksidatif stres olduğunu. Aynı deney, adapte edilebilir ve ROS kaynakları veya ROS üreten bileşikler, H2O 2 ve rotenone gibi kullanılabilir. Benzer deneyler, PQ düşük konsantrasyonlarda 25,26 kullanılan plakalar üzerinde geliştirilmiştir. Bu deneyde avantajı çok hızlı ve sonuçlar bir gün içinde elde edilebilir olmasıdır. Buna ek olarak, 96 gözlü plakalar içinde oksidatif stres direnci deneyi gerçekleştirmek için kullanılan sıvının toplam hacmi PQ-ihtiva eden plakalar hazırlamak için kullanılır hacmi ile karşılaştırıldığında düşüktür. Bu nedenle, kullanılan PQ miktarı sıvı analizde ise ucuz tahlil işler ve zehirli atıkların üretimini sınırlayan, düşük. Bununla birlikte, levha tahlilleri ile karşılaştırıldığında bu analizde sınırlamaları la bulunmaktadırSıvı deneyde gıda ve sıvı oksijen daha düşük bir konsantrasyonda CK hava ile karşılaştırıldığında. Bunlar bazı durumlarda, sonuçlar etkileyebilecek önemli faktörlerdir. Bu nedenle, bu tahlilde elde edilen sonuçlar desteklemek için tavsiye edilir, oksidatif stres direnci diğer yöntemlerle tekrarlanabilirlik teyit etmektedir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Agar bacteriological grade</td>
			<td>Multicell</td>
			<td>800-010-LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological peptone</td>
			<td>Oxoid</td>
			<td>LP0037</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride biotechnology grade</td>
			<td>Bioshop</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8503-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure tris hydrochloride</td>
			<td>Invitrogen</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris aminomethane</td>
			<td>Bio Basic Canada Inc</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-202185A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bioshop</td>
			<td>69-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Bio Basic Inc.</td>
			<td>8013-01-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl viologen dichloride hydrate</td>
			<td>Aldrich chemistry</td>
			<td>856177-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60x15mm</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10ml</td>
			<td>Fisher</td>
			<td>1367520</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>G-Biosciences</td>
			<td>RC-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>M-5921</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Bioshop</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Discovery v8 stereo zeiss microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well clear microtiter plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>flcn-1 RNAi source</td>
			<td colspan="2">Ahringer Library</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring oxidative stress resistance of caenorhabditis elegans in 96-well microtiter plates

Üretim ve reaktif oksijen türlerinin detoksifikasyon arasındaki dengesizliğin sonucudur Oksidatif stres, kardiyovasküler ve nörodejeneratif hastalıklar, diyabet, yaşlanma ve kanser gibi kronik insan bozuklukları, büyük bir katkıda bulunuyor. Bu nedenle, hücre sistemlerinde değil, aynı zamanda bütün organizmalar kullanılarak sadece oksidatif stres çalışma için önemlidir. C. elegans yüksek evrimsel korunmuş olan oksidatif stres sinyal iletim yollarının, genetiğinin incelemek için cazip bir model organizmadır. Burada, biz C. oksidatif stres direnci ölçmek için bir protokol sağlamak sıvı içinde elegans. Bu tür paraquat (PQ) ve H2O 2 Kısaca, ROS-indükleyici reaktifler M9 tampon içinde çözülür ve çözeltiler 96 gözlü bir mikrotitre plakasının gözenekleri içinde bölünür. Senkronize L4 / genç yetişkin C. elegans hayvanlar oyuklara transfer edilir (5-8 hayvan / oyuk) ve hayatta kalma ölçülürher saat en fazla solucanlar ölene kadar. Verilerin yanlış yorumlanmasına yol açabilecek oksidatif stres üzerine hayvanların davranışlarını etkileyebilecek yaşlanma, plakalarda streslerinin düşük konsantrasyonu kullanılarak bir oksidatif stres direnci deneyi yaparken. Bununla birlikte, bu tarifnamede tarif edilen deneyde, bu sorun yalnızca L4 / genç yetişkin hayvanlar kullanılmaktadır çünkü pek mümkün değildir. Ayrıca, bu protokol, ucuz ve sonuçlar genetik ekranlar cazip bu teknik kılan bir gün içinde elde edilmektedir. Genel olarak, bu oksidatif stres ile ilişkili insan bozukluklarının daha iyi karakterizasyonu tercüme edilebilir oksidatif stres sinyal iletim yollarını, anlamanıza yardımcı olacaktır.

Karşılaştırılması vahşi tip C. elegans FLCN-1 (ok975) mutant hayvanlar Burada yabani tip C direncini belirlemek için 100 mM PQ kullanılan oksidatif stres, ısı ve anoksi 23 karşı gösterilmiştir FLCN-1 (ok975) ile karşılaştırıldığında elegans hayvanlar. Tedavi 4 saat, yabani tip% 48.3 sonra hayatta olarak FLCN-1 (ok975) hayvanlarda% 77.8 hayatta kalma ile karşılaştırıldığında. Beklendiği gibi, FLCN-1 (o...

Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Oksidatif Stres Direnci Ölçüm<em&gt; Caenorhabditis elegans</em&gt; 96 oyuklu mikrotiter plakalarında

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

measuring-oxidative-stress-resistance-caenorhabditis-elegans-96-well

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Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

15.3K Görüntüleme.  McGill University. C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates. 

Video: Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Ölçme Caenorhabditis elegans Katı Medya Ömrü

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Biyoloji

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

İzolasyon ve In vitro Etkinleştirme Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Ölçme Caenorhabditis elegans 96 Şey microtiter Tabaklar Ömrü

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Erken Embriyo Oluşumu Zaman atlamalı Mikroskopi Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice-Cap: Büyüyen Bir Yöntem Arabidopsis</emYüksek Verim Genotiplendirme için 96-iyi Tabaklar ve Domates Tesisleri>

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Temel<em&gt; Caenorhabditis elegans</em&gt; Yöntem: Senkronizasyon ve Gözlem

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Prostaglandin Ekstraksiyon ve Analiz<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Zebra balığı Embriyolar Oksidatif Stres Analizi

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

yetiştirme<em&gt; Caenorhabditis elegans</em&gt; Laboratuvarı Üç boyutlu

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Oksijen tüketimi oranlarında sağlam Caenorhabditis elegans ölçümü

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...