Biz filme katkılarından dolayı George Jarvis, Martha Bock ve Bette Hecox-Lea, Deniz Biyoloji Laboratuvarı müteşekkiriz.

Medya 1. Hazırlık Not: kullan tuzluluk 16.5 ppt (PSU) yarı seyreltilmiş Brujewicz yapay deniz suyu. Diğer yapay deniz suyu, aynı zamanda sık sık kültür Brachionus rotiferler 25,26 için kullanılır. <ol><li> 454 mM NaCI, ekleme 26 mM MgCI2, 27 mM MgSO 4, 10 mM KCI, damıtılmış su, 10 mM CaCl2 4.5 L (nihai hacim 5 L) olacaktır. Alternatif olarak, deiyonize seyreltme suyu yerine distile su kullanın. Diğer tüm tuzlan çözüldükten sonra CaCl2 ekleyin. </li><li> 0.48 M NaHCO 3 stok çözelti (200x konsantrasyon) hazırlayın. Yukarıdaki çözeltiye bunun 25 ml ekleyin. NaHCO 3 nihai konsantrasyon 2.4 mM&#39;dir. </li><li> 0.4 M NaBr stok solüsyonu (500x konsantrasyon) hazırlayın. Yukarıdaki çözüm için 10 ml ekleyin. NaBr nihai konsantrasyonu 0.8 mM&#39;dir. damıtılmış su ile 5 litreye tamamlanır sağlayın. </li><li> 0.45 um membran filtre ile çözelti filtre.Kullanımdan önce, steril su ile iki kez seyreltilmiş (h / h). Not: Bir stok çözeltisi olarak Brujewicz yapay deniz suyu 2x konsantrasyonunu yapmak mümkündür. </li></ol> 2. Genel Kültür Koşullar <ol><li> Kültür laboratuar yükseltilmiş veya 20 ile 30 ° C arasında bir steril 100 ml çanak içinde yabani ele Rotiferler. Yüksek sıcaklıklar daha kısa ömürleri ve hızlandırılmış üreme sonuçlanır. Deneylerin kolaylık sağlamak için, 25 ° C ile. Rotifer yoğunluğu veya yapay deniz suyu hacmi burada büyük bir sorun değil. </li><li> Yapay deniz suyu 11 Kültür diyet mikroalg türleri. Snell ve ark bakın. (2014) Detaylar 11. Tipik haliyle, Tetraselmis tetrathele kullanımı (~ 2 x 10 5 hücre / ml 27,28), T. Suecica (~ 6 x 10 5 hücre / ml 25,29), ve Nannochloropsis oculata türü (~ 7 x 10 6 hücre / ml 30,31). diyet yosun beri (türler, kültür conditiyonları, biyokimyasal kompozisyonlar) önemli ölçüde tüm deney gruplarında mikroalgi aynı çok kullanırım, Brachionus rotifer yaşam süresini etkiler. </li><li> Beslenme ve (örneğin, 2 günde beslemek ve her hafta aşılamak) bazen medyayı değiştirerek bir toplu kültüründe rotifer bir stok nüfusu tutun. rotifer optimal koşullar olduğunda birçok yeni doğanlar ve 2-3 yumurta taşıyan yetişkinler görülebilir. Not: Deneysel rotifer elde etmek için alternatif bir yol dinlenme yumurta yumurtadan etmektir. Dinlenme yumurtalardan Rotiferler iyi senkronize olarak kabul ve onların ömrü amictic yumurta 11 yumurtadan rotifer önemli ölçüde farklı değildir vardır. Ancak, dinlenme yumurtadan rotiferler önce amictic yumurta daha üreme başlar. Böylece, dikkatli üreme özelliklerinin ölçümleri için gereklidir. </li></ol> Ön kültür ile Rotiferler 3. Senkronizasyon <ol><li> st tek bir rotifer seçinock nüfusu ve kültürü deneyleri için kullanılacak bir alt popülasyonu kurmak için 2.1-2.3 tarif edilmiştir. iki hafta içinde tipik olarak kültür. </li><li> alt nüfustan yumurta taşıyan rotifer toplayın (deney için kullanılacak bireylerin çift sayıda toplamak). Tek bir grup olarak kültür bunları (yoğunluk: ~ 50 birey / ml) ad libitum besleme altında taze ortam içinde 2.1-2.3 tarif edildiği gibi bir 6-yuvalı kültür plakasına. Koşullandırılmış ortam Brachionus rotifer 32,33 üreme fizyolojisini etkiler çünkü nüfus yoğunluğu kontrol eden önemlidir. </li><li> Yeni hazırlanan kültür ortamına yetişkinlerin ilk yumurtalardan yenidoğanlar aktarın. 2-3 nesil boyunca bu işlemi tekrarlayın. </li><li> Hayatta kalma süresini ölçmek için belirli bir süre (örneğin, &lt;3 saat) içinde yumurtadan yenidoğanlar kullanın. , Inde birkaç kullanarak tekrarlanabilirlik kontrol çok imkansız olmasına rağmen, tek bir birey seçiminde olası bir önyargı önlemek içinsarkık alt popülasyonları. Not: rotifer genellikle bu koşul altında sadece eşeysiz üreme olarak, hiçbir erkek deney boyunca mevcut olduğundan emin olun. Erkekler yenidoğan daha küçüktür ve genellikle hızlı kadınlarda daha taşımak. Mictic kadın, belirli koşullar altında 29 amictic kadın farklı ömürleri vardır. </li></ol> Survival of Time 4. Ölçümler <ol><li> Plastik levha yerleştirin yenidoğan (yapay deniz suyu içeren her 1 ml tipik haliyle 24 ya da 48 oyuklu plakalar). </li><li> 24 saat aralıklarla, eğer daha yüksek bir sıcaklıkta (30 ° C&#39;nin altında OR) yeni hazırlanmış kültür ortamı ya da 12 saatlik aralıklarla rotifer aktarın. döl sayısını kaydetmek ve her bireyin ölü ya da diri olup olmadığı. Koronanın kirpikler hareketi tamamen durduğunda ölü olarak rotifer kaydedin. Not: Rotiferler genellikle kuyu yan yüzeyine yapışırlar. Suyun yumuşak pipetleme onları bulmak için yardımcı olur. rotifer bulunamadı veya ar değilsenize yanlışlıkla &quot;sansürlü&quot; olarak değil &quot;ölü&quot; olarak, bunları kayıt, pipetle zarar. </li><li> Deneysel rotifer aktif üreyen zaman yenidoğanlar çıkarın. Yenidoğanlar hızla büyümeye ve deneysel rotifer onları ayırmak için bazen zordur. </li></ol> 5. Veri Analizi <ol><li> Kaplan-Meier sağkalım eğrisi (Şekil 1 ve 2) X ekseninde Y ekseni ve zamanında kümülatif sağkalım oranı çizerek oluşturun. Bu yaşam en yaygın veri temsilidir. Hayatta kalma süresi 34 istatistiksel karşılaştırma için (aynı zamanda Mantel-Cox testi denir) non-parametrik log-rank testi kullanın. log-rank testi aynı zamanda JMP ve R. gibi diğer standart istatistik paket dahildir Not: Normal dağılım genellikle hayatta kalma verileri 35 tarafından karşılandı çünkü Student t-testi veya parametrik çoklu karşılaştırma ardından varyans (ANOVA) analizi kullanmayın. Ayrıca, bu metoDS dikkate sansür bireyleri yapmayız. hiçbir sansür veri olup olmadığını Mann-Whitney U testi kullanılabilir. </li></ol>

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W., Johnston, R. K., Rabeneck, B., Zipperer, C., Teat, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Joint+inhibition+of+TOR+and+JNK+pathways+interacts+to+extend+the+lifespan+of+Brachionus+manjavacas+(Rotifera).">Joint inhibition of TOR and JNK pathways interacts to extend the lifespan of Brachionus manjavacas (Rotifera).</a> Exp. Gerontol. 52, 55-69 (2014).</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+nematode+Caenorhabditis-elegans+-+major+biological+and+environmental-factors+influencing+life-span.">Aging in nematode Caenorhabditis-elegans - major biological and environmental-factors influencing life-span.</a> Mech. Ageing Dev. 6 (6), 413-429 (1977).</li><li>Priest, N. K., Mackowiak, B., Promislow, D. E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+parental+age+effects+on+the+evolution+of+aging.">The role of parental age effects on the evolution of aging.</a> Evolution. 56 (5), 927-935 (2002).</li></ol>

The authors have nothing to disclose.

Mevcut protokolü Brachionus rotiferler sağkalım süresini ölçmek için bir yöntem tarif eder. Kritik adım birkaç nesil boyunca rotifer koşulları eşitleme olduğunu. Deneysel rotifer iyi senkronize edildiğinde birkaç önceki çalışmalarda 18,24,37,38 belirtildiği gibi, tipik bir tip I sağkalım eğrisi çok az erken mortalite ile görülmektedir. onların hayatta kalma süresi standart sapmaları dolayısıyla yüksek istatistiksel güç sonuçlanan kötü senkronize rotifer göre daha küçük olur. Senkronizasyon da yaşam süresi ölçümlerin tekrarlanabilirliği artması bekleniyor - anneler optimal koşullarda kültüre, çünkü mevcut protokol anne nesillerin olası zararlı etkilerini dengeler. Erken mortalite hala dikkatli senkronizasyon sonra gözlenirse, yeni hazırlanan kültür ortamı kullanmayı düşünün, yani beslenme yosun veya yeni kurulan deneysel kohort başka çok () Protokolü 3.1 başlayacak. Bu protokolün bir sınırlama iyi senkronize rotiferler potansiyel aşırı duyarlı olmasıdır. Örneğin, ömrünü uzatmak kimyasalların tarama üzerine, bu protokol ile taranmıştır bazı kimyasallar kötü senkronize rotifer ömürleri üzerinde önemli etkileri tespit etmek için başarısız olabilir (örneğin, vahşi ve toplu kültürlü toplumlarda bireylerin). Dolayısıyla, bu tür deneylerin sonuçları dikkatle yorumlanmalıdır. Yavru hayatta kalma süresi üzerinde anne yaşının etkisi de meyve sinek Drosophila melanogaster ve Caenorhabditis elegans 39,40 nematod dahil olmak üzere diğer omurgasız modellerinde bildirilmiştir. daha zaman alıcı olan bu uzun ömürlü bir model olan, ancak, bu hayvanların yaşam süresi ölçümü, deneysel varyasyon azaltmak için, birkaç nesil boyunca eşitleme işlemi yararlı olacaktır.

Rotiferler filumunu Rotifera 1 oluşturduğu mikroskopik kozmopolit zooplankton (&lt;1 mm) &#39;dir. Bu yaklaşık 1000 somatik hücreler gibi karakteristik bir tekerlek gibi siliyer cihazından oluşan basit bir vücut düzeni hareket kabiliyeti ve besleme için kullanılan korona, adı vardır. Çoğu rotifer yaklaşık 1.600 ve 500 tür, sırasıyla 2 ihtiva sınıflar Monogononta veya bdelloidea aittir. Bdelloid rotifer zorunlu parthenogenesis 3 ile yeniden oluşturma sırasında Monogonont rotiferler genellikle hem eşeyli ve eşeysiz üreme evreleri (döngüsel parthenogenesis) var. Deneylerde, yüksek tekrarlanabilirliği sağlanır genetik olarak özdeş rotifer bireyler elde etmek mümkündür. Buna ek olarak, bu kadar kısa bir ömrü, kültür kolaylığı, genomik ve transkriptomik dizi verilerine 4-7 kullanılabilirliği ve uzak benzersiz bir filogenetik pozisyon gibi model organizmalar gibi birçok başka avantajları varrthropods ve nematodların 8. Rotifer Bu nedenle ekolojik, toksikolojik omurgasız modelleri vaat ve çalışmalar 9-12 yaşlanma vardır. Çevresel strese ya da kimyasallara maruz altında hayatta kalma süresi bu araştırma alanlarında 13-19 bir çok ölçülen parametredir. Bu annelerinin çevre koşullarına maruz olduğu rotiferler hayatta kalma süresini ölçme, ancak, dikkatli gereklidir. Yani, monogonont rotiferi Brachionus manjavacas olarak, yaşlı annelerden gelen kadın yavrular genç annelerden daha kısa bir ömrü var; Ancak, anne kalori kısıtlaması (CR) kısmen ileri anne yaşı 20 zararlı etkilerini dengeler. B. plicatilis, anne CR yavru uzun ömürlü, açlık altında uzun yaşam süresi ve antioksidan enzimler 21,22 gelişmiş ifadesi ile ilişkili yüksek oksidatif stres direnci sağlar. anne yaşı etkisiAyrıca bdelloid rotiferler 23 gözlenmiştir. Bu nedenle, deneysel rotifer koşulları dikkatlice sağkalım süresi ölçümleri önce birkaç nesil boyunca senkronize edilmelidir. Burada birkaç nesil boyunca kültür koşulları senkronizasyon aşağıdaki Brachionus rotifer sağkalım süresinin ölçümü için bir protokol sağlar. Aralıklı açlık (IF), rotiferler periyodik beslenir CR bir varyasyon nedeniyle uzun ömürlü 22,24 tarihinde IF tanınmış etkilerine senkronizasyon etkisini ortaya çıkarmak için uygulanmıştır.

<table><tbody><tr><td>Sodium chloride</td><td>Wako</td><td>190-13921</td><td></td></tr><tr><td>Magnesium chloride</td><td>Wako</td><td>136-03995</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Wako</td><td>131-00427</td><td></td></tr><tr><td>Potassium chloride</td><td>Wako</td><td>168-22111</td><td></td></tr><tr><td>Calcium chloride</td><td>Wako</td><td>035-00455</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Wako</td><td>199-05985</td><td></td></tr><tr><td>Sodium bromide</td><td>Wako</td><td>190-01515</td><td></td></tr><tr><td>Membrane filter (0.45 &amp;micro;m pore size)</td><td>Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>Culture plate, 6-well, non-treated</td><td>Thomas Scientific</td><td>6902D01</td><td>Flat bottom</td></tr><tr><td>Culture plate, 48-well, non-treated</td><td>Thomas Scientific</td><td>6902D07</td><td>Flat bottom</td></tr><tr><td>Tetraselmis, Living</td><td>Carolina Biological Supply Company</td><td>152610</td><td></td></tr><tr><td>PRISM 6</td><td>GraphPad Software</td><td>Version 6.0d</td><td></td></tr></tbody></table>

measurement of survival time in brachionus rotifers: synchronization of maternal conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Şekil 1 kötü senkronize popülasyonlar (iki çoğaltır dışarı) temsilcisi yaşam eğrilerini göstermektedir. Bu deneyde, rotiferler vardı ya beslenen günlük [ad libitum (AL) grubu] veya (grup IF) her gün. Medyan sağkalım sırasıyla gruplar AL ve IF 13 ve 18 gün idi. iyi IF rotifer ömrünü uzatır olduğu bilinmesine rağmen, bu deney AL ve gruplar IF ömürleri arasında istatistiksel olarak anlamlı bir fark tespit edemedi. Ampirik, erken ölüm ve hayatta kalma oranı kademeli düşüşler yetersiz eşitleme sonuçları olarak bu deneyde gözlenen. alt nüfus için uygun olmayan tedavi veya düşük su kalitesi nedeniyle rotifer hasar benzer sonuçlar elde etmek eğilimindedir. Rotiferle koşulları uygun ve iyi senkronize olduğunda, erken mortalite pek gözlenen ve accordin olduğugly rotifer deney (Şekil 2) sonraki aşamasında senkronize bir şekilde ölmek eğilimindedir. Medyan sağkalım sırasıyla gruplar AL ve IF 13 ve 20 gün idi. Daha az hayvan Şekil 1&#39;de deney daha kullanılmasına rağmen, bu gruplar arasındaki ömrü fark istatistiksel olarak anlamlı idi. Bu daha önce 22 yayımlanmıştır beşten fazla deneyler temsilcisi sonuçları olduğunu. <img alt="Şekil 1" src="/files/ftp_upload/54126/54126fig1.jpg" /> Şekil 1:. EĞER grup her geçen gün beslendi ise aralıklı oruç (IF) tabi kötü senkronize bireyler için Kaplan-Meier eğrileri AL grubu, deney boyunca libitum beslendi. N AL için 11 ve n = 12 = ve Grupları sırasıyla (N deneyde kullanılan tek tek sayısını ifade eder). Deney performan- edildiMed, 25 ° C &#39;de. Log-rank testi (p = 0,1207) kullanıldığında ömrü açısından anlamlı bir fark saptanmadı. Ancak, bu veri testi 36 sağlam olması bilinmesine rağmen log-rank testi iki kapısı sağkalım eğrileri karşılaştırmak için kullanılmamalıdır çünkü yorumlamak zordur. Hiçbir kurulan yöntemleri şu anda sansürlü verilerle yaşam eğrileri geçiş için kullanılabilir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/54126/54126fig2.jpg" /> Şekil 2:. IF tabi senkronize bireyler için Kaplan-Meier eğrileri öncesi kültüründen elde edilen rotifer kohort, (her gün beslenen) aynı IF programa tabi tutuldu. N = 6 ve AL N = 8 ve gruplar IF, sırasıyla. Deney, 25 ° C &#39;de gerçekleştirildi. Log-rank testi, p = 0,0057. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions at JoVE.com

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Hayatta kalma Süre deneyinde<em&gt; Brachionus</em&gt; Rotiferler: Maternal Koşullarının Senkronizasyon

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

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Nanyang Technological University

Research

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Biology

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

8.4K Görüntüleme.  The University of Tokyo. Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Video: Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

Yineleme kümesi yöntemi: Bir yüksek-den geçerek yaklaşım kantitatif ölçü Caenorhabditis elegans ömrü

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Genetik

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical data that may be used in predictive modelling to forecast evolutionary responses of natural ecosystems to future environmental changes. However, excluding a few exceptional cases, long-term studies are scarce because of logistic difficulties associated with accessing temporal samples. Temporal dynamics are frequently studied in the laboratory or in controlled mesocosm experiments with exceptional studies that reconstruct the evolution of natural populations in the wild.
Here, a standard operating procedure (SOP) is provided to revive or resurrect dormant Daphnia magna, a widespread zooplankton keystone species in aquatic ecosystems, to dramatically advance the state-of-the-art longitudinal data collection in natural systems. The field of Resurrection Ecology was defined in 1999 by Kerfoot and co-workers, even though the first attempts at hatching diapausing zooplankton eggs date back to the late 1980s. Since Kerfoot&#39;s seminal paper, the methodology of resurrecting zooplankton species has been increasingly frequently applied, though propagated among laboratories only via direct knowledge transfer. Here, an SOP is described that provides a step-by-step protocol on the practice of resurrecting dormant Daphnia magna eggs.
Two key studies are provided in which the fitness response of resurrected Daphnia magna populations to warming is measured, capitalizing on the ability to study historical and modern populations in the same settings. Finally, the application of next generation sequencing technologies to revived or still dormant stages is discussed. These technologies provide unprecedented power in dissecting the processes and mechanisms of evolution if applied to populations that have experienced changes in selection pressure over time.

Resurrection of Dormant Daphnia magna: Protocol and Applications

Long-term studies are essential to understanding the process of evolution and the mechanisms of adaptation. Generally, these studies require commitments beyond the life-time of researchers. Here, a powerful method is described that dramatically advances state-of-the-art data collection to generate longitudinal data in natural systems.

Diriliş uyuyan Daphnia magna: Protokolü ve uygulamaları

Long-term studies enable the identification of eco-evolutionary processes that occur over extended time periods. In addition, they provide key empirical ...

Çevre

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 ...

Biyoloji

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

Caenorhabditis elegans'ta Yaşam Süresini ve Sağlık Süresini Ölçmek için Düşük Maliyetli Yöntemlerin İncelenmesi

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as self-fertilizing hermaphrodites, which produce large broods of progeny-when males are present, they can produce even larger broods of cross progeny. Errors in meiosis, fertilization, and embryogenesis can be rapidly assessed as phenotypes of sterility, reduced fertility, or embryonic lethality. This article describes a method to determine embryonic viability and brood size in C. elegans. We demonstrate how to set up this assay by picking a worm onto an individual Modified Youngren's, Only Bacto-peptone (MYOB) plate, establish the appropriate timeframe to count viable progeny and non-viable embryos, and explain how to accurately count live worm specimens. This technique can be used to determine viability in self-fertilizing hermaphrodites as well as cross-fertilization by mating pairs. These relatively simple experiments are easily adoptable for new researchers, such as undergraduate students and first-year graduate students.

Measuring Embryonic Viability and Brood Size in Caenorhabditis elegans

Here, we present a general method to determine the embryonic viability and total number of embryos produced (brood) using the model organism C. elegans.

Caenorhabditis elegans'ta embriyonik canlılık ve yavru büyüklüğünün ölçülmesi

Caenorhabditis elegans is an excellent model organism for the study of meiosis, fertilization, and embryonic development. C. elegans exist as ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

Pinewood Nematodu gözlem ve Çiftleşme Davranış miktarının belirlenmesi,<em&gt; Orijinli olanlar</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

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 ...

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater mussels are among the most imperiled organisms in the world, but information about early larval (glochidial) development and brooding periods is still lacking for many species. Previous studies have focused on the complex life history stage when female mussels are ready to parasitize host fish, but few studies have focused on the brooding period and timing of larval development. The protocol described here allows researchers to non-lethally evaluate the state of gravidity for female mussels. The results of this study show that this method does not affect a female mussel&rsquo;s ability to stay gravid or become gravid again after sampling has been performed. The advantage of this method may permit its use on federally threatened or endangered species or other populations of high conservation concern. This protocol can be adapted for use on both preserved or live individuals and was tested on a variety of mussel species. The database provided is a repository for a breadth of information on timing of reproductive habits and will facilitate future freshwater mussel research, conservation, and recovery efforts.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels Bivalvia: Unionida

Freshwater mussel conservation is dependent on monitoring reproductive patterns and processes of species. This study standardizes a non-lethal protocol for sampling gill contents, characterizing larval development, and providing a digital repository for data collected. This protocol-database package will be an important tool for mussel researchers in recovery of imperiled species.

Tatlı Su Midyelerinin Üreme Durumunu ve Larval Gelişimini Karakterize Etmek Için Ölümcül Olmayan Bir Yöntemin Standartlaştırılması (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Eleganlarda Fizyolojik Stres Tepkilerinin Ölçüleri

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Nematode Synchronization: A Method to Obtain Populations of Worms in Identical Stages of Development

Source: Sarasija, S. and Norman, K. R. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59277/measurement-oxygen-consumption-rates-intact-caenorhabditis">Measurement of Oxygen Consumption Rates in Intact&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp. (2019).
In this video, we show a method of producing a developmentally synchronized population of sterile adult C. elegans. The example protocol synchronizes worms to study oxygen consumption rates in live animals.

Nematod Senkronizasyonu: Aynı Gelişim Aşamalarında Solucan Popülasyonları Elde Etmek İçin Bir Yöntem

Source: Sarasija, S. and Norman, K. R. Measurement of Oxygen Consumption Rates in Intact&#160;Caenorhabditis elegans.&#160;J. Vis. Exp. (2019).
In this ...

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