Bu çalışma Gerontoloji Ödülü ve NIH Hibe MKGS 1R01AG031108-01, Ulusal Sağlık Enstitüsü, eğitim hibe P30AG013280 tarafından desteklenen bir Glenn / Afar buluşlar tarafından desteklenmiştir. MK Yaşlanma Ellison Tıp Vakfı Yeni Scholar.

Bölüm 1: hazırlayın nematod büyüme ortamı (NGM) plakaları. Bu bölümde, ömrü deneyde kullanılmak üzere sağlam NGM plakaların hazırlanmasında açıklamaktadır. Ampisilin (Amp) ve NGM eklendi fluorodeoxyuridine (FUDR) hiçbir katkı maddesi ve Amp / FUDR tabak, içeren standart NGM tabak, temel bir ömrü deney iki tip plakalar gerektirir. Ampisilin yabancı bakteri bulaşmasını önlemek için kullanılır. FUDR, hücre bölünmesini inhibe yumurta üretimini azaltır, ve kuluçkalık yumurta engeller. Uzun ömürlü analiz için FUDR yetişkin kullanım ömrü etkileyecek ve birkaç gün büyüyen larva onları ayırmak için her solucanları aktarmak için gereksinimini ortadan kaldırır. Her iki tür plakaların E. ile seeded sonradan UV ışınlarına maruz tarafından öldürülen coli OP50 bakteri. <ol><li> NGM (tarifi ve depolama notları için bkz: Bölüm 5) ve etiket petri tabak hazırlayın. NGM 10 ml başına 1 60 mm petri tabak gerekir. Önceden hazırlanmış katı NGM ile başlayan iseniz, 1.2 adıma geçin. Taze otoklavlanmış NGM ile başlayan iseniz, 1.4 adıma geçin. </li><li> Tamamen katı NGM 30 saniyelik bakliyat yüksek mikrodalga eritebilir. Kadar bina basınçlı gaz kabarcıklarını engellemek için bakliyat arasındaki Swirl medya. </li><li> 55 ° C su banyosu ya da soğutmak için bankta Yeri sıvı NGM. </li><li> Bir kez NGM ulaştığında 55 ° C, 150 mM FUDR 33 mcL ve 100 mg / ml, 100 ml NGM başına ampisilin (Amp / FUDR plakalar için) ve karıştırın girdap 100 mcL ekleyin. NGM plakaları için ek ilaçsız, 1.5 adıma doğrudan geçin. </li><li> Her 60 mm petri tabak içine steril tekniği, pipetle 10 ml NGM kullanma. Mümkünse kabarcıklar oluşturan kaçının. Kabarcıkları varsa, bunlar bir pipet kullanarak attı. </li><li> NGM katılaşmaya ve kuru izin kapaklarını bankta plakaları bırakın. Mümkünse, bakteri eklemeden önce 2 gün boyunca bankta plakaları bırakın ama plakaları er ihtiyaç vardır 1 gün çalışacaktır. </li><li> Günü plakaları E. taze bir çizgi tek bir koloni ile kurutma, tohum sıvı LB kültür bitirmeden önce LB agar coli OP50 bakteri. 60 mm plaka başına bir gecede OP50 kültürünün en az 1 ml hazırlamalıdır. </li><li> 37 Yeri OP50 kültür ° C çalkalayıcı ve bakteriler (kültür doygunluk ulaşmak olmalıdır) gece boyu büyümeye izin verir. </li><li> Pelet OP50 10 dakika boyunca 3.500 g iplik bakteri. </li><li> Süpernatant ve tekrar süspansiyon bakteri kültürü 10x konsantre% 90 çıkarın. </li><li> Pipet 100 mcL 10x konsantre OP50 kültür merkezi haline katılaşmış NGM plakalar. (Ideal bakteriler plaka duvarları yakınında gelmeden NGM merkezi alanı kapsamalıdır) gerekirse Swirl plakaları yavaşça bakteri kültürü yaymak için. Medya yüzey kusurları NGM içine yuva solucanlar izin verdiği ölçüde, NGM yüzeye pipet dokunmaktan kaçınmak için deneyin. </li><li> Bakteri kültürü katı NGM (genellikle 24 saat sürer) üzerinde kurumasına izin gecede bankta plakaları bırakın. </li><li> Kuruduktan sonra, plakaların yüzey bakterilerin büyüme durması için yeterli bir UV dozu maruz. Bir Stratagene UV Stratalinker 2400 kullanıyorsanız: <ul><li> Yeri Stratalinker plakaları ve kapakları kaldırmak </li><li> Kapıyı kapatın ve Stratalinker açmak </li><li> Basın &#39;Enerji&#39; </li><li> &#39;9999 &#39;Tuş takımını kullanarak girin </li><li> Press &#39;Başlat&#39; </li><li> Plakalar yaklaşık 5 dakika süreyle UV ışınlarına maruz kalacağı </li></ul></li><li> UV-öldürülmüş bakteri ile Tabaklar, 4 ° C 1 aya kadar baş aşağı saklanabilir. </li></ol> Bölüm 2: zamanlanmış bir yumurta hayvanların yaş senkronize bir nüfus elde etmek için döşeme gerçekleştirin. Bu bölümde ortak bir ambar güncel solucanları bir nüfusa üretir. Bu üreme aktif yetişkin için zaman belirli bir süre için bir plaka üzerine yumurtalarını bırakmak için izin ve bu yumurtaları geliştirmek için izin gerçekleştirilir. <ol><li> (Isteğe bağlı), yaklaşık 20 genç erişkin solucanlar FUDR olmadan yeni bir NGM plaka aktarın. 25 ° C, solucanlar, tüm bakteri gıda yoluyla yaymak ve yemek sağlamak amacıyla plakaları bırakın. Yeni yumurtadan çıkan solucanlar bakteriyel gıda tüketilen edildikten sonra tutuklanan büyüme Dauer sahne girecek. Başlangıçta plaka transfer kaç solucanlar bağlı olarak, bir hafta içinde yaklaşık Dauer larva görmeye başlayacaksınız. Dauer larva 1 ay kadar bir sonraki adımlar için kullanılabilir. </li><li> FUDR olmadan taze NGM plaka (İsteğe bağlı) Transfer 20 30 Dauer larva. , 2 gün içinde üreme aktif yetişkin gıda Dauer larva varlığı haline gelecektir ve bundan sonra 25 üreme birkaç gün boyunca aktif kalır ° C </li><li> 10 ila 15 üreme aktif yetişkin FUDR olmadan yeni bir NGM plaka aktarın. Bu plaka Zamanlı yumurtlama (TEL) plaka olarak adlandırılır. </li><li> 25 ° C 6 saat için izin vermek için TEL plaka bırakınsolucanlar zaman yumurtalarını bırakmak için. </li><li> TEL plaka erişkin solucanlar çıkarın. Plaka yetişkin çıkarmadan önce yumurta için görsel olarak kontrol edilebilir. TEL plaka, solucanlar, yeterli sayıda yumurta koydu varsa erişkin solucanlar çıkarmadan önce 24 saat olmak üzere toplam 25 ° C kadar sol olabilir. </li><li> Place TEL plaka 20 ° C yumurtadan yumurta var ve solucanlar L4 larva dönemi (bu genellikle vahşi tip C. elegans için 2 gün sürer, ama yavaş gelişimi fenotipleri suşları için uzun sürebilir) için geliştirdiği kadar. </li></ol> Bölüm 3: Puan hayvanların ömrü Ölünceye kadar bu bölümde Bölüm 2 solucanlar yaş senkronize nüfus izleyin. Worms, yumurta üretimi ve bakteriyel kontaminasyonu önlemek için Amp / FUDR tabak tutulur ve dış uyaranlara yanıt vermeyen ölü olarak kabul edilir. <ol><li> Numaralı seribaşı Amp / FUDR plakaları L4 larva transferi. Her bir gerginlik ya da koşulu test için, plaka başına 25-30 solucanlar ile 2-3 tabak kurmak için tipik bir örneğidir. C. Görüntüler elegans yaşam evrelerinde referans verilmektedir (Şekil 1) 1 </li><li> Place Amp / FUDR plakaları 20 ° C 24 saat boyunca. </li><li> 24 saat sonra, görsel, solucanlar, medya, ve bakteri değerlendirmek. Aşağıdakilerden herhangi birini görülmektedir taze Amp / FUDR plakaları solucanlar transferi: <ul><li> Worms bakteriyel gıda yemiş. Erken, ömrü deney solucanlar tükenmiş olan bakterileri önlemek için bir hafta 1 ila 2 kez transfer edilebilir olacaktır. </li><li> Larvaların önemli bir kısmı mevcuttur. Bu genellikle solucanlar L4s yerine genç yetişkinler olarak Amp / FUDR plakaları ve FUDR yürürlüğe girmiştir önce bazı yumurtalarını bırakmak için mümkün olduğu bir göstergesidir. FUDR genellikle tam yetişkin içine büyüyen larva önlemek, ancak bazen bir kaç yetişkin içine büyümek ve deney hayvanları ile karıştırılmamalıdır olacak. </li><li> Canlı / büyüyen bakteriler gözlenir. Genel olarak, Amp ve UV-öldürme arada hiçbir canlı bakteri deney kontamine sağlayacaktır, ancak zaman zaman ortaya çıkar. </li><li> Mantar büyüme medya görülmektedir. Yeterince erken yakalanmış ise, mantar üremesine genellikle bir pipet veya spatula kullanarak kesip olabilir. Bir kere, birkaç milimetre çapında yeni bir plaka solucanlar aktarmak için genellikle daha kolaydır daha büyük olması yetişir. </li><li> Diseksiyon kapsamı kullanarak, her solucan, canlı ya da ölü olup olmadığını belirlemek. </li><li> Plaka hafifçe dokunun. Solucan dinlenmesine tepki olarak hareket ederse canlıdır. </li><li> Solucan başkanı bölgeye dokunarak levha, zoom için yanıt vermezse. </li><li> Platin transfer mızrapla solucan kafasını hafifçe dokunun. Puan başını hareket ettirerek yanıt vermemesi durumunda ölü olarak solucan. </li><li> Ölü solucanlar plaka çıkarılabilir. </li></ul></li><li> Canlı ve ölü bir tarih ve solucanlar sayısını kaydedin. </li><li> 20 ° C&#39;ye kadar plakaları dön </li><li> Tüm solucanların ölmüş kadar tekrarlayın 3.3 3.6 sayesinde her 2 ila 3 gün adımları. </li></ol> Bölüm 4: Temsilcilik Sonuçları. Bir nematod ömrü deneyi tarafından üretilen ham veri, numaralarını içeren test her bir suş için canlı ve ölü solucanları tarihleri ​​bir liste. Her gün ölen sayısı solucanlar genellikle, bir hayatta kalma eğrisi (Şekil 2B olarak grafiksel olarak çizilen her gün canlı solucanlar oranı (Şekil 2A), hesaplamak için ters; Zamanlı yumurtlama günü 0. gün olarak kabul edilir .) Çalışmada her bir solucan için ömrü, her gün ölen solucanlar sayısı hesaplanır ve suşları arasında karşılaştırma için ortalama ve medyan yaşam süresi hesaplamak için kullanılabilir. Her gün canlı solucanlar sayısı sayımı ömrü analizi doğrudan kullanılmaz. Worms katı ortam üzerinde muhafaza bazen &#39;kaçmak&#39;, ya da medya altındaki plaka veya aşağı duvar ya tarama. Her gün canlı solucanlar deney kurs boyunca pek çok solucan, nasıl kaçtı sayısını belirlemek için kullanılabilir. Kaçmak Solucanlar genellikle analiz kaldırılır. N2, C. için bir kriter olarak, tipik bir medyan yaşam süresi 20 UV-öldürülmüş bakteri muhafaza elegans yabani tip gerilim ° C yumurtadan ölçülen yaklaşık 25 gündür. Bölüm 5: Çözümler <table border="0"><tbody><tr><td colspan="2"> Nematodu Büyüme Medya (NGM), 100 ml: </td></tr><tr><td colspan="2"> Birleştirin: </td></tr><tr><td> 0,3 g </td><td> NaCl </td></tr><tr><td> 0.25 g </td><td> Pepton </td></tr><tr><td> 2 g </td><td> Agar </td></tr><tr><td colspan="2"> 40 dakika otoklav ve soğuması ile 55 ° C, sonra ekliyorum: </td></tr><tr><td> 100 mcL </td><td> 1 M MgSO 4 </td></tr><tr><td> 100 mcL </td><td> 5 mg / ml Kolesterol </td></tr><tr><td> 100 mcL </td><td> 1 M CaCl2 </td></tr><tr><td> 1.625 ml </td><td> 1.5 M KPI pH 6.0 </td></tr><tr><td colspan="2"> Sıvı NGM oda sıcaklığında plakaları dökmek için hemen kullanılabilir veya sağlamlaştırmak için izin ve uzun vadeli saklanır. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> Luria Broth (LB), 1L: </td></tr><tr><td> 10 g </td><td> BactoTryptone </td></tr><tr><td> 5 g </td><td> Maya Ekstraktı </td></tr><tr><td> 10 g </td><td> NaCl </td></tr><tr><td> 10 ml </td><td> 1 M Tris pH: 8.0 </td></tr><tr><td> 1 L </td><td> deiyonize su </td></tr><tr><td colspan="2"> Otoklav ve oda sıcaklığında saklayın. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M MgSO 4, 300 mL: </td></tr><tr><td> 73,95 g </td><td> MgSO 4 </td></tr><tr><td> 300 mL </td><td> deiyonize su </td></tr><tr><td colspan="2"> Otoklav ve oda sıcaklığında saklayın. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 5 mg / ml Kolesterol, 200 mL: </td></tr><tr><td> 1 g </td><td> kolesterol </td></tr><tr><td> 200 mL </td><td> % 100 etanol </td></tr><tr><td colspan="2"> Sterilize Filtre ve oda sıcaklığında muhafaza. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M CaCl 2, 500 ml: </td></tr><tr><td> 27,75 g </td><td> CaCl 2 </td></tr><tr><td> 500 mL </td><td> deiyonize su </td></tr><tr><td colspan="2"> Sterilize Filtre ve oda sıcaklığında muhafaza. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1.5 M KPI pH 6.0, 1L: </td></tr><tr><td colspan="2"> Birleştirin: </td></tr><tr><td> 31.4 g </td><td> KPI dibazik </td></tr><tr><td> 179,6 g </td><td> KPI tekbazlı </td></tr><tr><td> 850 mL </td><td> deiyonize su </td></tr><tr><td colspan="2"> Isı KPI çözüm çözünmesi için karıştırma sırasında. 10 N NaOH ile pH 6,0 &#39;a ayarlayın. </td></tr><tr><td colspan="2"> 1 L. deiyonize su ekleyin </td></tr><tr><td colspan="2"> Otoklav ve oda sıcaklığında saklayın. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M Tris, pH 8.0: </td></tr><tr><td> 60,57 g </td><td> Tris </td></tr><tr><td> 400 mL </td><td> deiyonize su </td></tr><tr><td colspan="2"> HCl ile pH 8.0 ayarlayın. </td></tr><tr><td colspan="2"> 500 mL deiyonize su ekleyin. </td></tr><tr><td colspan="2"> Sterilize Filtre ve oda sıcaklığında muhafaza. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 150 mM Fluorodeoxyuridine (FUDR), 10 ml: </td></tr><tr><td> 0,3693 g </td><td> FUDR </td></tr><tr><td> 10 ml </td><td> steril deiyonize su </td></tr><tr><td colspan="2"> -20 ° C&#39;de muhafaza </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 100 mg / ml ampisilin (Amp), 10 ml: </td></tr><tr><td> 1 g </td><td> Ampisilin </td></tr><tr><td> 10 ml </td><td> steril deiyonize su </td></tr><tr><td colspan="2"> -20 ° C&#39;de muhafaza </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 50 mg / ml Carbenicillin (Carb), 10 ml: </td></tr><tr><td> 500 mg </td><td> Carbenicillin </td></tr><tr><td> 10 ml </td><td> steril deiyonize su </td></tr><tr><td colspan="2"> -20 ° C&#39;de muhafaza </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M İzopropil β-d-Thiogalactopyranoside (IPTG), 10 ml: </td></tr><tr><td> 2.38 g </td><td> IPTG </td></tr><tr><td> 10 ml </td><td> steril deiyonize su </td></tr><tr><td colspan="2"> -20 ° C&#39;de muhafaza </td></tr></tbody></table><img src="/files/ftp_upload/1152/Figure%201.png" alt="Şekil 1" /> Şekil 1. C. Aydınlık alan görüntüler yumurta da dahil olmak üzere elegans yaşam evreleri, dört larva aşamaları (L1-L4) ve yetişkin. Tüm paneller, yetişkin bir erkek (Wood (1988) Image), sağ alt hariç hermafroditlerde göstermektedir. <img src="/files/ftp_upload/1152/Figure%202.png" alt="Şekil 2" /> C. Şekil 2 Temsilcisi sonuçları elegans ömrü deneyi daf-2 genindeki bir mutasyon içeren bir yük vahşi tip suşu N2 karşılaştırarak. (A) toplanan solucanların yumurtaları beri gün sayısı da dahil olmak üzere veriler, her gün görülen ölü solucanlar sayısını, ve her gün hayatta kalan orijinal numunenin yüzdesi (ölü solucanlar günlük sayıları hesaplanan) gösteren bir tablo . (B) Survival ve (A) ömrü verilere karşılık gelen eğriler. <imgsrc = &quot;/ files/ftp_upload/1152/Figure 3.jpg&quot; alt = &quot;Şekil 3&quot; /&gt; Şekil 3 genç ve yaşlı yetişkin C arasında lipofusin Temsilcisi karşılaştırma elegans. Aydınlık alan görüntülerin sağ ve sol DAPI kanal floresan görüntüler gösterilir. En paneller 4 gün eski bir solucan ve alt paneller (yumurta ölçülen gibi) 11 gün eski solucan göstermektedir.

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+luminescence+spectroscopy+of+fluorescence+changes+during+aging+in+Caenorhabditis+elegans.">Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans.</a> Biochemistry. 21, 4089-4095 (1982).</li><li>Klass, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+the+nematode+Caenorhabditis+elegans:+major+biological+and+environmental+factors+influencing+life+span.">Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span.</a> Mech Ageing Dev. 6, 413-429 (1977).</li><li>Steinkraus, 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=Dietary+restriction+suppresses+proteotoxicity+and+enhances+longevity+by+an+hsf-1-dependent+mechanism+in+Caenorhabditis+elegans.">Dietary restriction suppresses proteotoxicity and enhances longevity by an hsf-1-dependent mechanism in Caenorhabditis elegans.</a> Aging Cell. 7, 394-404 (2008).</li></ol>

Uzun ömürlü genetik kontrol C. yaygın olarak incelenmiştir elegans, büyük ölçüde ömrü tespit edilebilir olan kolaylığı ve hızı nedeniyle. Bu makalede açıklanan protokol C. tekrarlanabilir ömrü veri elde etmek için temel bir çerçeve tanımlar elegans ve relatednematode türler için de uygulanabilir. 2, bu işlemler çok çeşitli şartlar altında ömrü ölçmek için adapte edilebilir basit bazı değişiklikler yaparak . Burada RNA enterferans (RN...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Agar</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0145-17-0 (214530)</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>MidSci</td>
<td>0339</td>
<td>&nbsp;</td>
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<td>BactoTryptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0123-17-3 (211705)</td>
<td>&nbsp;</td>
<tr>
<td>BactoPeptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0118-17-0 (211677)</td>
<td>&nbsp;</td>
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<td>CaCl2</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9699248 (1332-01)</td>
<td>&nbsp;</td>
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<td>Carbenicillin</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>C0109</td>
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<td>Cholesterol</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>C75209</td>
<td>&nbsp;</td>
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<td>Fluorodeoxyuridine (FUDR)</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F0503</td>
<td>&nbsp;</td>
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<td>Isopropyl &#x03B2;-d-Thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>I2481C5</td>
<td>&nbsp;</td>
<tr>
<td>KPi Dibasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087862 (3252-01)</td>
<td>&nbsp;</td>
<tr>
<td>KPi Monobasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087861 (3246-01)</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9561800 (2500-01)</td>
<td>&nbsp;</td>
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<td>NaCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S251</td>
<td>&nbsp;</td>
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<td>Tris</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
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<td>Yeast Extract</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0886-17-0 (288620)</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring caenorhabditis elegans life span on solid media

Yaşlanma, hücresel bileşenleri ve ölüm ile sonuçlanan organelleri ilerleyici bir bozulma ile karakterize dejeneratif bir süreç. Nematod<em&gt; Caenorhabditis elegans</em&gt; Yaşlanma biyolojisi okumak için kullanılan bir ana model olarak ortaya çıkmıştır. Hemen hemen her biyolojik alt sistemi, artan yaş ile fonksiyonel düşüş uğrar Çünkü yaşlanma toplam oranı göz önüne alındığında, ömrü ilgi birincil son nokta. Nematod, ömrü genellikle bir hayvan dış uyaranlara yanıt kalır gün sayısı olarak tanımlanır. Nematodlar ya da sıvı ortamda veya plakalar katı ortam üzerinde yayılır olabilir ve her şartlar altında ömrü ölçüm teknikleri geliştirilmiştir. Burada katı nematod büyüme ortamı devam nematod ömrü ölçmek için genelleştirilmiş bir protokol mevcut ve UV-öldürülmüş bakteri bir diyetle beslenen. Bu prosedürler kolayca canlı bakteri, diyet kısıtlaması, ve RNA girişim oluşan bir diyet de dahil olmak üzere, çeşitli ortak koşullar altında tahlil ömrü adapte olabilir.

Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Life Span on Solid Media at JoVE.com

Measuring Caenorhabditis elegans Life Span on Solid Media

Bu yazıda, UV öldürüldü bakteriyel gıda ile sağlam bir medya üzerinde muhafaza nematod ömrü ölçmek için genel bir protokol mevcut.

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 ...

measuring-caenorhabditis-elegans-life-span-on-solid-media

Research

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Biology

Measuring Caenorhabditis elegans Life Span on Solid Media

29.7K Görüntüleme.  University of Washington. Bu yazıda, UV öldürüldü bakteriyel gıda ile sağlam bir medya üzerinde muhafaza nematod ömrü ölçmek için genel bir protokol mevcut.

Video: Measuring Caenorhabditis elegans Life Span on Solid Media

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

Yaşlı Hücre akıbet tarafından Maya Kronolojik Ömrü miktarının

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

Biyoloji

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Geliştirici Maya replikatif Ömrü Ölçüm

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

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 ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Katı Plaka tabanlı Diyet Kısıtlama Caenorhabditis elegans</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

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 ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

Kültürleme<em&gt; Caenorhabditis elegans</em &gt; Mikroparçacık bombardımanı ile aksenik Sıvı Medya ve Transgenik Worms Yaratılış

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

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

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

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 ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Dokuya Özgü Chaperone Etkileşimlerini Taramak için Caenorhabditis elegans'ı Kullanma

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Uyarılmış Raman Saçılma Mikroskopisi Kullanılarak Caenorhabditis elegans Lipid Depolama Dinamiklerinin Etiketsiz Görüntülenmesi

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

Caenorhabditis elegans Health Üzerinde Etkisi Olan Mikrobiyal İzolatların Yüksek Verimli Taraması

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

Methods Article

Ölçme Caenorhabditis elegans Katı Medya Ömrü