Biz S. Taneja-Bageshwar, K. Beifuss, S. Kedroske, ve HC teşekkür etmek istiyorum. Yararlı tartışmalar için Hsiao. Bu çalışma, Moleküler Tıp TAMHSC Anabilim Dalı start-up fonları tarafından finanse edildi. Bileşik kapsamı ve dönen disk departmanı ve TAMHSC Dekan Ofisi tarafından sağlanan fonlar ile satın alındı. Bazı suşlar, Ulusal Araştırma Kaynakları Merkezi tarafından finanse edilen Caenorhabditis Genetik Merkezi tarafından verilmiştir . pRF4 (rol-6 (su1006)) A. Ateş bir hediye oldu.

1. Hazırlık DII, leke <ol><li> DMF 20 mg / ml DII (Biotium, Inc, Hayward, CA) stok solüsyonu hazırlayın. DII ışığa karşı hassastır, bu yüzden folyo sararak ışık DII korumak. </li><li> 0.6 mcL DII stok her nüfus için 399,4 mcL M9 ekleyerek DII bir çalışma seyreltme oluşturun. Bu son bir çalışma seyreltme 30 mcg / ml DII M9 vermelidir. Bu, aynı anda birden fazla popülasyonlar boyama için ölçeklendirilebilir. Kalkan DII tüp (ler) folyo sararak ışık. </li></ol> 2. Nematod hazırlanması <ol><li> Kirlenmemiş nematod bir nüfusu içeren 60 mm levha kullanın. Tüm larva ve yetişkin hayvanların yavaşça gevşetin plaka yüzey üzerinde dairesel bir hareket içinde sıvı dönen bir çözüm% 0.5 Triton X-100 M9 tampon kullanarak plaka hayvan yıkayın. 1,5 mL steril bir tüpe transfer yıkayın. </li><li> 2000 rpm&#39;de 30 sn için hayvanların hemen aşağı doğru döndürün. Kadar destek çıkartın ve atıntüp altındaki hayvanların kitle bozmadan mümkün olduğunca ernatant. </li><li> Azaltmak için artık Triton X-100, M9 tampon, spin kullanarak hayvanların temizleyin, durulayın ve süpernatant kaldırmak. Bir kez bu adımı yineleyin. </li><li> Çözüm hayvanların tekrar süspansiyon tüp ve vorteks kısaca DII çözüm M9 olarak çalışan 400 mcL ekleyin. </li><li> 20 tüp sallayın ° C yatay ışık korumalı bir ortamda 350 rpm&#39;de 3 saat. İsterseniz, hayvan boyama için 16 saate kadar inkübe edilebilir. </li><li> Bağlanmamış boya miktarını azaltmak için, 2000 rpm&#39;de 20 sn için hayvanların aşağı doğru döndürün. Çıkartın ve hayvanların kitle rahatsız etmeden, mümkün olduğunca çok supernatant olarak atın. </li><li> 400 mcL M9 tampon ve OP50 E. numaralı seribaşı NGM agar plaka bir bakteri kısmı üzerine sıvı dökün yeniden süspanse hayvanlar coli. Hayvanların en az 30 dakika kurtarmak için izin ver. Iyileşme süresi sırasında hayvanlar DII boyama sıvı ve gıda üzerine sürünerek gerekir. Bu adımücretsiz DII arka floresan azaltır. </li></ol> 3. Montaj ve gözlemleyerek örneklerin <ol><li> Bir otoklav veya mikrodalga kullanarak su içinde% 4 agar eritin. </li><li> Yeniden kullanılabilir ayırıcılar, bir bardak slayt laboratuvar bant katman iki adet agar pad tek tip kalınlık sağlamak için kullanılabilir oluşturun. Toplam iki ayırıcı slaytlar olun. </li><li> Iki ayırıcı slaytlar arasında temiz bir cam slayt düzenleyin. % 4, temiz bir cam slayt merkezi üzerine erimiş agar Pipet yaklaşık 150 mcL (dört damla). Hızla erimiş agar agar pad oluşturmak için ek bir slayt kullanarak kapsar. Montaj slayt üst merkezli ped dikkatlice tutarak, kapağını kaydırarak çıkarın. </li><li> Pipet nematod anestezik (100 mcM - 1 mM levamizol, örneğin) 5 mcL hakkında ped üzerine. </li><li> Dağı 8 - 12 anestezi hayvanlar ve bir mikroskop lamel ile kaplayın. </li><li> En az bir 40x objecti ile donatılmış bir bileşik veya konfokal mikroskop kullanılarak hayvanlar dikkat<html> inceledim ve TRITC / DSRed (veya diğer uyumlu) filtresi. DII floresans uyarma maksimum 549 nm ve emisyon bağlı boya (Biotium, Inc Hayward, CA) için maksimum 565 nm. </li></ol> 4. Temsilcisi Sonuçlar Yabani tip ve mutant C. DII lekeler manikür elegans. Cuticular yüzey annuli bazı aşamalarında, çevresel oluklar ile ayrılmış ve içeriyorsa, çatılar alae çağırdı. Her bir gelişim aşaması, 2 farklı kompozisyonlar ile cuticular yapılara sahiptir . Alae ve annuli hem de dişler veya oluklar, larva ve yetişkin aşamalarında, floresan yüzey bileşimi bağlı olarak, leke ve bu yöntemi kullanarak bir gün sonra kurtarma görünür kalır. Arkaplan floresan benekler bazen (Şekil 2F, G), ancak rutin olarak değil (Şekil 1, Şekil 2A-E, H, I). Tüm görüntüler kaydetti dönen disk konfokal veya, widefield (wf) bileşik mikroskobu ile alınmıştır. ent &quot;&gt; <img alt="Şekil 1" src="/files/ftp_upload/3362/3362fig1.jpg" /> Şekil 1. DII lekeler manikür ve çevre maruz kalan nöronlar. L2 aşamasında hayvan. 630x büyütme. Mozaik görüntü parçaları ivision-Mac yazılımı (BioVision Teknolojileri, Exton, PA) kullanılarak ele geçirildi. Görüntüler, Adobe Photoshop CS3 (, Adobe Systems, Inc, San Jose, CA) ile katıldı. Ölçek çubuğu = 10 mm. DII da floresan lekeleri amphid ve baş ve kuyruk phasmid duyusal nöronlar sırasıyla (oklar bazı işareti). <img alt="Şekil 2" src="/files/ftp_upload/3362/3362fig2.jpg" /> Şekil 2. DII floresan lekeleri C. post-embriyonik gelişimin her aşamasında elegans manikür. 630x büyütme. Ölçek çubuğu = 10 mm. A) L1 vahşi hayvanların Boyama B) L2 C) Dauer; D) L3; E) L4 ve F) yetişkin aşamaları. Alae ve halkalı sırtlar floresan (A, C), L1 ve Dauer hayvanlar lekeli. L2 hayvanlar DII lekeleri anüler sırtlar(B). Anüler, L3 ve L4 hayvanlar (D, E) leke oluklar. Alae oluklar ve annuli yetişkin hayvanların (FH) lekeli. Alae hayvanın uzunluğu (oklar) 16 çalıştırmak, iki, beş, ya da üç sırtlar (L1, Dauer, sırasıyla veya yetişkin hayvanlar,) oluşmaktadır . Annuli hayvan etrafında dairesel sırtlar (ok ucu, F ve J) oluşturun. Manikür yetişkin mutant hayvanlar orta cuticular organizasyon defektleri (GH) gösterir. G) alae, Ridges süreksiz (wf). H) supernumerary alae sırtlar erimiş ve dallı ya da çatallı (wf). Kollajen gen mutantlar alae ve anüler organizasyon defektleri (Aİ) sergilerler. I) transgenik hayvanlar eksprese pRF4 (rol-6 (su1006)), hayvanın uzunluğu bir açıyla alae yalan sırtlar. Transgenik hayvanlar eksprese pRF4 (rol-6 (su1006)) J) Annuli düzensiz bir desen gösterir. <img alt="Şekil 3" src="/files/ftp_upload/3362/3362fig3.jpg" /> Şekil 3. Eksternal morfolojik yapıları DII boyama aydınlatılmıştır. 630x büyütme. Ölçek bar = 10 mm. DII A) yetişkin hermafrodit vulva, B) yetişkin erkek kuyruk ışınları ve fan dahil olmak üzere diğer dış özellikleri, olayları, ve C) hermafrodit kuyruk başak (bu mutant arka planda çatallı). <img alt="Şekil 4" src="/files/ftp_upload/3362/3362fig4.jpg" /> Şekil 4. Tırnak çevreye maruz kalan nöronlar daha DII leke daha uzun sürer. 630x büyütme. Ölçek çubuğu = 10 mm. Iki saat sonra boyama, amphid (gösterilmemiştir) ve phasmid duyusal nöronları (oklar) yeterince lekeli. Buna karşılık, genç hayvanların manikür sadece kısmen yamaları (ok ucu) lekeli. <table border="1"><tbody><tr><td> Yıkama </td><td> Leke çözüm (+ DII) </td><td> İnkübasyon süresi </td><td> Tırnak boyandı </td></tr><tr><td> M9 +% 0.5 Triton X-100 </td><td> M9 +% 0.5 Triton X-100 </td><td> 2 saat </td><td> yok </td></tr><tr><td> M9 +% 0.5 Triton X-100 </td><td> M9 +% 0.5 Triton X-100 </td><td> 3 saat </td><td> yok </td></tr><tr><td> M9 +% 0.5 Triton X-100 </td><td> M9 </td><td> 2 saat </td><td> kısmi </td></tr><tr><td> M9 +% 0.5 Triton X-100 </td><td> M9 </td><td> 3 saat </td><td> evet </td></tr><tr><td> M9 +% 0.5 Triton X-100 </td><td> H 2 0 </td><td> 2 saat </td><td> kısmi </td></tr><tr><td> M9 +% 0.5 Triton X-100 </td><td> H 2 0 </td><td> 3 saat </td><td> evet </td></tr></tbody></table> Tablo 1. Farklı koşullar altında cuticular boyama Çeşitli kuluçka çözümleri ve saatleri hayvanlar cuticular boyama optimize etmek için test edildi. H 2 0, steril distile su. Kısmi boyama rağmen yetişkin manikür lekeleri c, larva manikür yamalı boyaması (Şekil 4) gösterironsistently.

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Biz ifşa etmek başka bir şey var.

Burada sunulan DII boyama yöntemi C. elegans manikür görselleştirmek için oldukça hızlı ve rahat bir şekilde sağlar . Repurposing ve optimize ederek, yaygın olarak görüntü çevreye maruz duyusal nöronlar 15,17 kullanılan bir yöntemdir, DII floresan alae ve halkalı yapılar (Şekil 1 ve 2), yanı sıra vulva, erkek kuyruk ve hermafrodit kuyruk başak leke için kullanılabilir (Şekil 3). Biz DII yeteneği inkübasyon çözüm ve zaman etkisi sürekli manikür (Tablo 1, Şekil 4) leke bulduk. DII boyama çevreye maruz kalan nöronlar için yöntem, Triton X-100 15 M9 iki saatlik bir inkübasyon süresi kullanır . Sürfaktan ile M9 bir ön yıkama, Triton X-100 cuticular yüzey kirlenme temizlenmesine yardımcı olur ve topaklanma gelen hayvanlar önler. Ancak, aynı tampon boyama çözüm hayvanların üç saat inkübe manikür (Tablo 1) boyama boya engeller.Bu lipidler, deterjan solüsyonu uzun tedavi çıkardı nematod yüzey üzerinde neden olabilir. M9 tuz solüsyonu manikür DII boyama için gerekli olmadığını belirten DII lekeler manikür su hayvanların Kuluçka. Nöronal DII boyama 15 su bazlı bir protokol olmasına rağmen, hayvanların bu şekilde çoğu zaman sağlıksız görünür tedavi bulmak . Boyama çözüm M9 ile yapılan üç saatlik bir inkübasyon tarafından takip Triton M9 X-100,% 0.5 kısaca hayvanlar Yıkama cuticular yapıları tutarlı boyama sağlar ve hayvan refahını korumaktadır. Hayvanlar gözlem sonra kurtarıldı ve doğrudan akış aşağı analizleri izin, bakımlı olabilir. Bu yöntem de dahil olmak üzere, pek çok çalışmada kullanılacak uygun bir araç, ancak, cuticular sekresyonu ve organizasyon, epidermal hücre gelişimi, heterochronic gen yollar ve nematod evrim ile sınırlı değildir.

<table border="1"><tbody><tr><td> Reaktifler </td><td> Eşanlamlılar </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> DII </td><td> 1,1 &#39;-Dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perklorat </td><td> Biotium, Inc. </td><td> 60010 </td><td> Stok seyreltme: DMF 20 mg / ml çalışma seyreltme: 30 mg / ml </td></tr><tr><td> DMF </td><td> Dimethylformamide </td><td> Sigma-Aldrich, Inc. </td><td> D4551 </td><td></td></tr><tr><td> Triton X-100 </td><td> Octylphenoxypolyethoxyethanol </td><td> VWR International, LLC. </td><td> EM-9410 </td><td></td></tr><tr><td> M9 </td><td> 22mm KH 2 PO 4, 42mm Na 2 HPO 4, 86mm NaCl, 1mm MgSO 4 </td><td></td><td></td><td></td></tr><tr><td> NGM &lt;/ Td&gt; <td> Nematodu büyüme ortamı </td><td> IPM Scientific, Inc. </td><td> 11006-501 </td><td> 18 NGM agar protokol hazırlanmış olabilir </td></tr><tr><td> Agar-agar </td><td></td><td> Merck Kimyasallar, Inc. </td><td> 1,01614 </td><td> % 4 su </td></tr><tr><td> Levamizol </td><td> Levamizol hidroklorür </td><td> Sigma-Aldrich, Inc. </td><td> 31742 </td><td> 100 mcM - 1 mM levamizol gerektiği gibi </td></tr><tr><td> Mikroskop slaytlar </td><td></td><td> VWR International, LLC. </td><td> 16005-106 </td><td></td></tr><tr><td> Mikroskop kapak camları </td><td></td><td> VWR International, LLC. </td><td> 16004-302 </td><td></td></tr><tr><td> Bileşik kapsamı </td><td></td><td> Carl Zeiss, Inc. </td><td> A1M </td><td> Deney ihtiyaçlarına uygun hedefleri kullanın </td></tr><tr><td> TRITC veya diğer uyumlu filtre </td><td> &amp;nbsp; </td><td> Chroma Technology Corp. </td><td> 49005 </td><td> ET - DSRed (TRITC/Cy3) filtre seti Püskürtülen </td></tr></tbody></table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

C. kütikül elegans hayvan 1-4 dış çevreler son derece dayanıklı bir yapıdır . Manikür hayvan çevreden korur, aynı zamanda vücut şekli belirler ve motilitesi 4-6 önemli bir rol oynar sadece. Epidermal hücreler tarafından salgılanan çeşitli katmanları bir dıştaki lipit tabaka 7 de dahil olmak üzere, manikür oluşmaktadır. Dairesel sırtlar manikür annuli desen hayvan uzunluğu olarak adlandırılır ve geliştirme 8 tüm aşamalarında bulunmaktadır. Alae Belirli gelişim aşamaları sırasında L1, Dauer ve yetişkin aşamaları 2,9 de dahil olmak üzere mevcut çatılar. Cuticular kollajen organizasyonu etkileyen genlerindeki mutasyonlar cuticular yapısı ve hayvan vücut morfolojisi 5,6,10,11 değiştirebilir. Cuticular yapıları Fluores, cuticular görüntüleme DIC optik ile bileşik mikroskobu kullanarak vurgulamak mümkün, güncel yöntemleryüzde transgen ifade 12, antikor boyama 13 ve elektron mikroskobu 1. Etiketli buğday tohumu agglutinin (WGA) cuticular glikoproteinler görselleştirmek için kullanılan, ancak daha ince cuticular yapılar 14 çözümünde sınırlıdır. Floresan boya kullanarak cuticular yüzey boyama gözlenmiş, ancak ayrıntılı 15 hiç karakterize olmuştur. Biz canlı C. manikür görselleştirmek için bir yöntem mevcut kullanarak elegans, yaygın olarak C. kullanılan kırmızı floresan lipofilik boya DII (1,1 &#39;-dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perklorat ) elegans çevreye maruz kalan nöronlar görselleştirmek için. DII boyama için optimize edilmiş Bu protokol, yüksek çözünürlüklü floresan annuli görselleştirme, alae, vulva, erkek kuyruk ve C hermafrodit kuyruk başak için basit, güçlü bir yöntemdir elegans.

Watch this Scientific Journal Video about Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI at JoVE.com

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Biz canlı manikür görselleştirmek için bir yöntem mevcut<em&gt; C. elegans</em&gt; Yaygın olarak kullanılan kırmızı floresan lipofilik boya DII (1,1 &#39;-dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perklorat)<em&gt; C. elegans</em&gt; Çevreye maruz kalan nöronlar görselleştirmek. Alae ve halkalı cuticular yapılar optimize edilmiş bu protokol ile, DII tarafından boyanmış ve bileşik mikroskobu kullanarak gözlenen.

Görselleştirme Caenorhabditis Elegans Tırnak Yapıları Lipofilik Vital Boya, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

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Nanyang Technological University

Research

JoVE Journal

Biology

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

17.0K Görüntüleme.  Texas A&M University System Health Science Center, College of Medicine. Biz canlı manikür görselleştirmek için bir yöntem mevcut C. elegans Yaygın olarak kullanılan kırmızı floresan lipofilik boya DII (1,1 '-dioctadecyl-3, 3,3', 3'-tetramethylindocarbocyanine perklorat) C. elegans Çevreye maruz kalan nöronlar görselleştirmek. Alae ve halkalı cuticular yapılar optimize edilmiş bu protokol ile, DII tarafından boyanmış ve bileşik mikroskobu kullanarak gözlenen.

Video: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

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 ...

Biyoloji

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

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

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

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

Vasküler Görselleştirme İçin Bir Yöntem Olarak DiI Perfüzyonu<em&gt; Ambystoma mexicanum</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Caenorhabditis elegans hücresel yapıları ve organelle morfoloji eğitim için nicel yaklaşımlar

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Arınma ve Caenorhabditis elegans Ekstrasellüler Veziküllerin Analizi

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

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, ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Caenorhabditis elegans'ta Agrega Niceliğinin Otomatikleştirilmesi

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Caenorhabditis elegans Genomik DNA'sının Küçük Ölçekli Ekstraksiyonu

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...