This research was supported by NIH Grant AG043080 and the National Natural Science Foundation of China (NSFC), No. 11434001. We thank Lucas Waldburger for proofreading the manuscript.

1. Silikon Gofret Kalıp İmalat NOT: Işık maskesi AutoCAD yazılımı ile tasarlandı ve ticari bir firma tarafından üretilmektedir. Bu tasarım farklı desenler (üç kat içerir <a href="http://ecsource.jove.com/files/ftp_upload/55412/single_cell_trap.dwg" target="_blank">Ek İçerik 1</a> ). Birinci, ikinci ve üçüncü tabakaların yükseklikleri sırasıyla yaklaşık 4 um, 10 um ve 50 um olan. Silikon gofret kalıp yumuşak litografi 9, 10 kullanılarak photomask oluşturuldu. <ol><li> 10 dk su buharı buharlaşması için 200 ° C&#39;da bir silikon gofret pişiriniz. Spin kat negatif fotorezist SU-8 silikon gofret üzerine. Not: Spin kat SU-8 3005, 30 ilk bir tabaka oluşturmak için 5000 rpm&#39;de, SU-8 2010 3000 rpm&#39;de 30 ikinci bir tabaka oluşturmak için, ve SU-8 3025 2,000 rpm&#39;de 30 saniye üretmek için üçüncü tabaka. </li><li> t önce kaplanmış gofret Yumuşak fırındaDesen transferini diye. Hizalayın ve bir maske hizalama kullanarak &quot;doğrudan temas&quot; modunda gofret maruz. Not: Yumuşak fırında birinci tabaka, 95 ° C&#39;de 2 dakika, ikinci tabaka 95 ° C &#39;de 3 dakika, ve üçüncü tabaka için 95 ° C&#39; de 15 dakika süre ile çok ince bisküvi. 100 mJ / Birinci tabaka için cm2, 130 mJ / ikinci katman için cm2 ve 200 mJ / üçüncü katmanın için cm2&#39;de maruz doz kullanın. </li><li> maruziyet sonrasında gofreti sonrası pişir ve SU-8 geliştirici kullanarak geliştirmek. N2 silah ve sert fırında 30 dakika 200 ° C &#39;de çok ince bisküvi ile gofret kurutun. Not: silikon gofret kalıp model tarafı yukarı bakacak şekilde, File bandı kullanılarak 15 cm çaplı plastik Petri kabına sabitlenmiştir. Genellikle, çok sayıda mikro-akışkan kartları aynı zamanda imal edilmesini sağlar aynı kalıp, birçok aynı çip yapıları koydu. Her bir kalıp mikroakışkan fiş imal etmek için pek çok kez yeniden kullanılabilmektedir. </li></ol> 2. mikroakışkanÇip Fabrikasyon <ol><li> Bir denge üzerinde temiz bir tartı tekne yerleştirin ve dengeyi daralayın. tartmak tekne içine PDMS tabanının 50 g dökün. </li><li> tartı teknesinde PDMS kürleme maddesi, 5 g ekleyin (ağırlık / PDMS tabanına 1:10 oranında ağırlık). NOT: Bu hacim, kalıp ile bir 15 cm çaplı bir Petri tabağına dayanır. Farklı büyüklükte bir petri kabı kullanarak, eğer reaktif miktarını ayarlayın. </li><li> PDMS tabanı ve tek kullanımlık bir pipet ile sertleştirme ajanı karıştırılır. tartım kabı kenarından başlayıp yavaş yavaş içeri doğru hareket eder. küçük kabarcıklar karışım boyunca meydana kadar birkaç dakika süreyle iyice karıştırılır; Karışımın tam PDMS polimerizasyonu için esastır. </li><li> yavaş Petri kabı içine dökün; Karışım, tam olarak silikon gofret kalıp kapsamalıdır. </li><li> 10 dakika PDMS karışımından tüm hava kabarcıklarını çıkarmak için bir vakum içinde Petri tabağına yerleştirin. kabarcıklar karışımın yüzeyinde kalır ise, onları dışarı üflemek için bir pipet kullanın. </li><li> silikon inkübeyaklaşık 2 saat süreyle 75 ° C&#39;de bir fırın içinde PDMS ile gofret kalıbı. </li><li> Yavaşça gofret kalıp yapımı için herhangi bir zarar vermeden, silikon gofret kalıp kapalı polimerize PDMS tabaka soyun. Seçenek olarak ise, bir tek kenarlı endüstriyel jilet kullanılarak desen etrafında en az 5 mm aralık ile silikon gofret kalıp şirketinden PDMS kesme; hafifçe gofret kalıp kapalı PDMS katmanı soyun. </li><li> model tarafı yukarı bakacak şekilde tezgah PDMS tabaka yerleştirin. Dikkatle yapı hasarı önlemek için her bir çip kenarından uygun bir devamlılığının bir tek kenar endüstriyel jilet ile tek tek çip kesip. </li><li> yukarı bakacak şekilde model tarafı, her bir kanal tarafına düz bir şekilde giriş ve çıkış çevrelerine boyunca delikler bir zımba kalem (0.75 mm İD) kullanın. NOT: Bu delikler ortamın akışı için yol oluşturun. Nedenle, çevrelerinde geçmesi ve PDMS tabakasının içinden tüm yol yumruk için çok önemlidir. Emin oldelikten PDMS sütunları kaldırın. </li><li> deliğe tekrar delme kalem iğne sokarak her delinmiş delik edin. İğne herhangi bir blokaj bulunmamaktadır belirten diğer taraftan dışarı gelebilir emin olun. Desen yüzeye bant uygulama yavaşça toz parçacıklarını temizlemek için bant soyulabilir. bu adımı en az üç kez tekrarlayın. sterilizasyonu korumak için PDMS üzerinde viski bant temiz bir parça bırakın. </li><li> PDMS&#39;den karşı tarafında bu işlemi tekrarlayın ve yanı üzerine bant son dilimini bırakın. </li><li> 0,13-0,17 mm kalınlığında, 24 mm x 30 mm kapak camı hazırlayın. yüzey sterilize etmek için toz sökücü ile cam üzerinde% 70 etanol püskürtme ve kurutma; buna ek olarak, cam otoklavlanmış su ile yıkandı ve toz sökücü ile kurutulabilir. </li><li> Plastik bir plakaya kapak cam ve PDMS aktarın. PDMS viski bandını çıkarın ve yukarı bakacak desen tarafını yerleştirin. Aktarım sırasında desen yüzeye sahip herhangi bir temastan kaçının. </li><li&gt; Plazma makinesinde plastik levha yerleştirin. PDMS oksijen plazma işlemi, aşağıdaki işlem parametrelerine sahip hidrofilik yüzeyler oluşturmak için cam kapak geçerlidir: maruziyet, 75 s; Gaz sabitleme, 20 cc / dakika; basınç, 200; ve güç, 100 W </li><li> Dikkatle hidrofilik yüzeyler (plazma işlemi sırasında karşılaştığı yüzeyler), bağlantı, kapak camı üzerine PDMS yerleştirin. PDMS ve kapak cam arasında hava kabarcığı olmadığından emin olun. </li><li> en az 2 saat boyunca 75 ° C&#39;de bir fırın içinde PDMS çip inkübe edin. Not: Eksik mikro sıvılaştırma deney sırasında mikroskop üzerine sızıntıya neden olabilir. Bu nedenle, biraz cımbız ile kenarlarından PDMS kaldırarak PDMS ve kapak camı arasındaki bağı kontrol edilmesi önemlidir. Yavaşça başarılı bağı belirten kapak camı ayırmak gerektiğini PDMS kaldırarak. </li></ol> 3. Deney hazırlığı <ol><li&gt; Tüp hazırlanması. <ol><li> % 70 etanol çözeltisi ayrıca PDMS çip hazırlanmasından önce 1 gün içinde giriş ve çıkış boruları daldırın. </li><li> tüpleri yıkamak için otoklava su ile 5 ml şırınga doldurun. şırınga üzerindeki tüp takmayı ve su ile tüp ateş basması her tüp yıkayın. </li><li> etanol kalıntısını temizlemek için, yıkama adımı en az 3 kez tekrarlayın. </li></ol></li><li> emin yapı, tutarlı ve sağlam olmak için 10X objektif ile bir optik mikroskop altında PDMS kanalları inceleyin. File bandı kullanılarak mikroskop platformu üzerine PDMS çip stabilize. NOT: İlk defa cihaz yapma, özellikle yedekleme cips var olmasını sağlamak için adım 2&#39;de tek seferde birden cips olun. </li><li> otoklava su ile 5 ml şırınga doldurun. bir şırınga pompası şırıngayı edin ve karşılık gelen giriş ve çıkış boruları yerleştirin. yaklaşık 10 dakika boyunca çip durulama 750 uL / ​​saat çamaşır hızını ayarlayın. şube kanallarında hava kabarcıklarıBu süre sırasında uzaklaşmış olmalıdır; Kalan kabarcıklar varsa, elle kabarcıklarını yok etmek için hızını ayarlamak. </li><li> Maya numune hazırlama. <ol><li> 3.000 x g&#39;de 5 dakika boyunca iki adet 1.5 ml mikrosantrifüj tüpü içine ml hazırlanan maya örneği (OD600 0.6-0.8) devri 1 ve santrifüj. </li><li> Her bir tüpten yüzer çoğunluğu çıkarın ve 0.5 ml örnek oluşturmak üzere geri kalan birleştirir. </li></ol></li><li> şırınga pompası Pause ve PDMS, çip giriş tüpleri kaldırmak. </li><li> El ile her bir giriş tüpüne (uzunluk olarak 1 ila 2 cm) küçük bir hava kabarcığı emmek için şırınga pompası ters. maya örneğinde emmek geçin; hava kabarcığı örnek sınırını göstermek ve çizilmiştir ne kadar örnek gösterecektir. NOT: şırıngaya örnek emme kaçının. </li><li> Lütfen PDMS çip içine giriş tüpü yerleştirin ve 750 uL / ​​saat bir hızda hücrelerin yüklenmesi için pompayı yeniden. </li><li> 10 dakika üzerinde pompa şırınga bırakınburada n ve mikroskop altında hücre yükleme ilerlemesini incelemek; direği askıya fazla% 60 altında başarılı bir yükleme olacak tuzak hücreleri. </li><li> beslenme solüsyonu geçin ve hücre kültürü için 400 uL / ​​saat hızı ayarlayın. </li><li> mikroskop için seçin gözlem pozisyonlar. NOT: ömrü ölçümleri için, görüntüler 40x amacı ile bir optik mikroskop ile her 15 dakikada alındı. flüoresan raportör analizi için, görüntüler 60X petrol amacı ile bir floresan mikroskobu ile bir kez, her 15 dakikada bir alındı. </li></ol>

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The authors declare that they have no competing financial interests.

PDMS cihazı taze yapılmış olması gerekmektedir. Aksi takdirde, cihaz içine tüpler sokulması neden olduğu hava kabarcıkları uzaklaştırmak için zor olacaktır. Adım 3.4 hücrelerini yoğunlaşarak hücre yükleme verimliliğini artırmak için önemlidir. Deneyin verimini arttırmak için aynı PDMS çip üzerinde 4 ila 6 modül bağımsız olarak çalışan pompalar genellikle eş zamanlı olarak 4 ila 6 farklı deneyler (farklı tür ya da ortam bileşimler) gerçekleştirmek için kullanılan bağlı. </...

mayayı tomurcuklanan araştırma yaşlanma güçlü bir model organizmadır. Bununla birlikte, mayada, geleneksel bir ömrü deneyi sadece emek yoğun değil, aynı zamanda, düşük verimlilik, 1, 2 olan mikrodiseksiyon dayanır. yaşlandıkça Ayrıca, geleneksel mikrodiseksiyon yaklaşım bekar bir anne hücrelerde çeşitli hücresel ve moleküler özelliklerinin ayrıntılı bir görünümünü sağlamaz. Mikro-akışkan cihazlarının gelişimi maya ömrü ölçmek için hem de ana hücreleri, 3, 4, 5, 6, 7, 8, ömrü boyunca moleküler işaretçileri ve çeşitli hücre fenotipleri takip otomatik bir prosedür sağladı. Maya hücreleri, bir mikro-akışkan cihazın içine yüklendikten sonra, bunlar otomatik zaman tur kullanılarak mikroskop altında izlenebilenE görüntüleme. Işleme araçları görüntülenmesi sayesinde, çeşitli hücresel ve moleküler fenotipleri, 3 elde edilebilir 6 kullanılarak elde edilmesi zor veya imkansız olan birçok vb ömrü, boyut, floresan haberci, hücre morfolojisi, hücre döngüsü dinamiği, dahil olmak üzere, 8, geleneksel mikrodiseksiyon yöntemi. Mikroakışkan cihazlar birkaç yıl önce 3, 4, 6, 7 başarılı gelişme beri maya yaşlanma araştırmalarında önem kazanmıştır. Çeşitli gruplar sonradan daha erken tasarımları 5 varyasyonları yayınlanan ve birçok maya laboratuarları kendi çalışma için microfluidic cihazlar kullanmışlardır. üstel büyümeyi geçiren bir hücre kültüründe, gözlem için mevcuttur yaşlı anne hücre sayısı miniscu olduğunule. Bu nedenle, ömrü ölçümler için mikro-akışkan cihazının genel tasarım prensibi anne hücreleri korumak için ve kızı hücreleri kaldırmaktır. Bu tür tasarımlar maya asimetrik hücre bölünmesini uğrar gerçeği kullanır. Cihaz irade kapanı büyük anne hücrelerinde yapı ve izin küçük kızı hücreleri uzakta yıkanacak. Bu makalede açıklanan mikro-akışkan çip tuzak ana hücrelere yumuşak polidimetilsiloksan (PDMS) ped (dikey sarkık sütun) (Şekil 1) kullanır. Benzer bir tasarıma sahip cihazlar daha önce 3, 4, 6, 7 bildirilmiştir. Bu protokol microfluidic cihazlar ve zaman atlamalı görüntüleme deneyleri için optimize edilmiş basit bir hücre yükleme yöntemi üretmek için daha basit bir prosedür kullanır. mikroakışkan cihaz önemli parametrelerden biri tuzak ana hücrelere kullanılan PDMS pedleri genişliğidir. Bizim dihaz hayatları boyunca izlenebilir taze anne hücrelerinin önemli bir kısmı dahil olmak üzere her yastık altında daha anne hücreleri tutabilir geniş yastıkları kullanır. Hücreler nesiller veya tüm ömrü boyunca bir gözlem gereklidir için izlenmesi gereken zaman ömrü ölçümlerine ek olarak, bu protokol, tek bir hücre zaman atlamalı görüntüleme deneyleri için kullanışlıdır.

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			<td>3&#39;&#39; &#60;111&#62; silicon wafer</td>
			<td>Addison Engineering</td>
			<td></td>
			<td></td>
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		<tr>
			<td>SU-8 2000 and 3000 Series</td>
			<td>MicroChem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD&#174; 184 SILICONE ELASTOMER KIT</td>
			<td>ellsworth</td>
			<td>2065622</td>
			<td>Include Sylgard&#174; silicone elastomer base and curing agent</td>
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		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>391-1502</td>
			<td></td>
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		<tr>
			<td>Harris Uni-core&#8482; punch(I.D. 0.75 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>29002513</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm x 40 mm SLIP-RITE&#174; cover glass</td>
			<td>Thermo Fisher Scientific</td>
			<td>102440</td>
			<td></td>
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			<td>3M&#160; Scotch Tape</td>
			<td>ULINE</td>
			<td>S-10223</td>
			<td></td>
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			<td>VWR&#174; Razor Blades</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
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		<tr>
			<td>PURE ETHANOL, KOPTEC</td>
			<td>VWR</td>
			<td>64-17-5</td>
			<td></td>
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			<td>WHOOSH-DUSTER&#8482;</td>
			<td>VWR</td>
			<td>16650-027</td>
			<td></td>
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			<td>5mL BD Syringe (Luer-Lock&#8482;Tip)</td>
			<td>Becton, Dickinson and Company.</td>
			<td>309646</td>
			<td></td>
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			<td>PTFE Standard Wall Tubing (100ft, AWG Size:22, Nominal ID: 0.028)</td>
			<td>COMPONENT SUPPLY COMPANY</td>
			<td>SWTT-22</td>
			<td></td>
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			<td>Needle Assortment</td>
			<td>COMPONENT SUPPLY COMPANY</td>
			<td>NEKIT-1</td>
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			<td>Desiccator</td>
			<td>HACH</td>
			<td>2238300</td>
			<td></td>
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			<td>Lab Oven</td>
			<td>FISHER SCIENTIFIC</td>
			<td>13246516GAQ</td>
			<td></td>
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			<td>Nikon TE2000 microscope with 40x and 60x objective</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
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			<td>Zeiss Axio Observer Z1 with 40x and 60x objective</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Longerpump</td>
			<td>TS-1B</td>
			<td></td>
		</tr>
	</tbody>
</table>

using microfluidic devices to measure lifespan and cellular phenotypes in single budding yeast cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

Deneylerden sonra, hücreler ve birçok hücresel ve moleküler fenotipleri yaşam süreleri kaydedilmiş zaman atlamalı görüntüleri elde edilebilir. her bir hücreden ekstre edilebilir, farklı bir dizi özellik olduğundan, analiz ilk adım, pozisyonları ve hücre sınırları ve izlenen çeşitli olayların zamanlama dahil olmak üzere hücreleri ve olayları, açıklama şekildedir tomurcuklanan olaylar olarak. Bu ek açıklamalar daha kolay gelecekte farklı özelliklere hücre...

Watch this Scientific Journal Video about Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells at JoVE.com

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Bu makalede, mikro akışkan cips üretim ve kullanım ömrü ve tek bir maya hücrelerinin hücresel fenotipleri ölçmek için mikro-akışkan deney kurulumu için optimize edilmiş bir protokol sunulur.

Mikroakışkan Aygıtlar Kullanma Tek Budding Maya Hücreleri ömrü ve Hücresel fenotipleri Ölçmek için

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

using-microfluidic-devices-to-measure-lifespan-cellular-phenotypes

Research

JoVE Journal

Biology

7.6K Görüntüleme.  Peking University.  Bu makalede, mikro akışkan cips üretim ve kullanım ömrü ve tek bir maya hücrelerinin hücresel fenotipleri ölçmek için mikro-akışkan deney kurulumu için optimize edilmiş bir protokol sunulur. 

Video: Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

Içinde Postembryonic Fenotiplerin Belirleme RNAi Tarama C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

In ömrü ölçümü<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Tomurcuklanma Maya Floresan Time-lapse Filmler Kazanılması ve GREFTLERİ kullanarak Tek hücreli Dynamics analiz

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

Tek Maya Hücreleri MAPK İsteyerek İfade zamansal Kantitasyonu

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Büyük ölçekli Gen demonte olarak<em&gt; C. elegans</em&gt; Sağlam Kayıp fonksiyon-fenotipleri oluşturmak için DsRNA Besleme Kitaplıklar kullanma

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Fonksiyon Çalışma, Etkileşimler, ve Post-translasyonel değişiklikler için tomurcuklanan Maya Protein Ekstraksiyonu ve Saflaştırılması

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Budding Maya endositozun kantitatif, kinetik ve Yüksek verim Analizi pHluorin Uygulamaları

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Fisyon Maya Cytokinetic Olayların zamanmekansal Analizi

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Yüksek çözünürlüklü görüntüleme ve Bireysel Astral Mikrotubul Dynamics Analizi Budding Maya

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...