Bu çalışma, A.-LH Nathan Şok Merkezi (P30 AG13283) ve Ulusal Yaşlanma Enstitüsü (NIA) R01 hibe (R01 AG028516) bir pilot ödül tarafından desteklenen

<html> 1. Bakteri konsantrasyonlarının tahmini için bir kalibrasyon eğrisinin oluşturulması Bu bölümde, C. Diyet kısıtlama deneyler için bir besin kaynağı olarak kullanılır, herhangi bir bakteri suşu (örneğin OP50, HT115) bakteri konsantrasyonu tahmini için bir yöntem açıklanır . elegans. Ayrı kalibrasyon eğrileri bakteri suşu kullanılan her tesis edilmelidir. <ol><li> E. tek bir koloni İnokülasyon coli OP50 bakteri, 3 ml sıvı LB suyu LB agar bakteri taze bir çizgi aldı . </li><li> 37 Yeri OP50 kültür ° C çalkalayıcı ve bakteri gecede büyümek için izin verir. </li><li> (2x, 4x, 8x, 16x, 32x) gece kültüründen bakteri süspansiyon seri 2 kat dilüsyonları hazırlayın. </li><li> Her seyreltme (optik yoğunluk) absorbansı 600 nm&#39;de spektrofotometre tarafından üç nüsha olarak ölçün. </li><li> Ayrıca her bakteriyel süspansiyon 1.000.000 kat (iki kez 1000 kat seyreltme) sulandırmak. </li><li> 100 mm LB agar plaka üzerine eşit olarak her seyreltme bakteriyel süspansiyon 0.4 mL yayıldı. </li><li> 18-24 saat boyunca 37 ° C inkübatör OP50 LB tabak yerleştirin. </li><li> Her plaka üzerinde yetişen bakteri kolonilerinin sayısını sayın. Koloni sayımları, her bir örnek için mL başına cfu (koloni oluşturan birim) hesaplamak için kullanılır. </li><li> Bakteriyel konsantrasyonu (kob / ml) bir fonksiyonu olarak OD 600 komplo ve lineer regresyon analizi (Şekil 1) performans ölçülen OD 600 değerleri ve OP50 bakteri konsantrasyonları arasındaki ilişki kurulabilir. </li></ol> Çözümler: <table border="1"><tbody><tr><td colspan="2"> Luria Broth (LB), 1L: </td></tr><tr><td > 10 g </td><td> Bacto-Tripton </td></tr><tr><td> 5 g </td><td> Maya Ekstraktı </td></tr><tr><td> 10 g </td><td> NaCl </td></tr></tbody></table> 1L deiyonize su ekleyin. Otoklav ve oda sıcaklığında saklayın. 2. Katı plaka tabanlı diyet kısıtlaması (SDR) plakalarının hazırlanması Bu bölüm katı plaka dayalı diyet kısıtlaması (SDR) deneylerde kullanılmak için plakalar hazırlanması açıklamaktadır. Normalde, düşünün bir bakteri konsantrasyonu 1 x 10 11 kob / ml ad libitum besleme durumu ve 1 x bu yöntem için en uygun DR beslenme koşulu olarak 10 8 kob / ml. Ancak, 4-6 farklı bakteri konsantrasyonları SDR ve yanıtları için tam bir doz-bağımlı bir ilişki kurmak için gerekli olabilir. <ol><li> E. tek bir koloni İnokülasyon 3 ml sıvı LB suyu Coli OP50 bakteri. </li><li> 37 ° C shaker&#39;da OP50 kültür koyun ve 6-8 saat bakteriler büyümek için izin. </li><li> 500 ml LB suyu içeren yeni bir balona OP50 kültür aktarım ve 37 ° C çalkalayıcı gecede geri OP50 kültürünü yerleştirmek. </li><li> Bakteri konsantrasyonu (konsantrasyonu Bölüm 1 oluşturulan kalibrasyon eğrisi kullanılarak hesaplanmıştır) belirlemek için OP50 kültürü OD 600 ölçün. Uygun dilüsyonları absorbans doğru bir ölçüm için gerekli olabilir. </li><li> 20 dakika 1500 g santrifüj Pelet OP50 bakteri. </li><li> 1 x 10 12 kob / ml &#39;lik bir konsantrasyon bakteriler tekrar </li><li> Altı farklı konsantrasyonlarda 10 ml bakteri kültürü hazırlayın: 1 x 10 12 10 11 10 10 10 9 10 8 10 7 kob / ml. </li><li> Pipet 0,2 ml 60 mm merkezi haline bakteri kültürü NGM plaka pekiştirdi. Yüzeyde nickler agar plakları içine yuva solucanlar izin, pipet ile plaka yüzeyine dokunmaktan kaçının. </li><li> 1 saat süreyle 37 ° C&#39;lik inkübatöre yerleştirin plakalar. </li><li> Bakterilerin büyüme tutuklamak için her plaka 100mg/mL carbenicillin ve 50mg/mL kanamisin 10 mcL 10μL uygulayın. </li><li> Mağaza plakaları 4 farklı konsantrasyonlarda antibiyotik öldürülmüş bakteri ° C ileride kullanmak için (bir ay kadar). Tüm planlanan deney için önümüzdeki en az 1-2 gün yetecek kadar tabak hazırlamak ve saklamak için çok önemlidir. Aynı parti tüm deneyler için plakaların kullanılması tercih edilir. </li><li> Bakterilerin canlılığını doğrulamak için, SDR plakaları bazı bakteri kazımak ve carbenicillin ve kanamisin boş bir NGM plaka üzerine yayıldı. 6-8 saat 37 ° C inkübatör plaka koyun ve daha sonra bakteri büyümesi için (hiç bakteri büyüme dikkat edilmelidir) kontrol edin. </li></ol> Çözümler <table border="1"><tbody><tr><td colspan="2"> Nematodu Büyüme Medya (NGM), 1L: </td></tr><tr><td > 3 g </td><td> NaCl </td></tr><tr><td> 2.5 g </td><td> Bacto-Peptonlu </td></tr><tr><td> 20g </td><td> Agar </td></tr></tbody></table>ove_content &quot;&gt; 40 dakika için Otoklav ve soğuması için 65 ° C, daha sonra ekleyin: <table border="1"><tbody><tr><td > 1 ml </td><td> 1 M MgSO 4 </td></tr><tr><td> 1 ml </td><td> 1 M CaCl 2 </td></tr><tr><td> 1 ml </td><td> 5 mg / ml Kolesterol </td></tr><tr><td> 25 ml </td><td> 1M potasyum fosfat, pH 6.0 </td></tr></tbody></table> 10 ml alikotları hemen 60 mm x 15 mm petri kutularının üzerine sıvı NGM dökün 3. SDR deneyler ayarlama Ömrü analizi ve doğurganlık profili, solucanlar yaş senkronize nüfus hazırlanması gibi SDR-ile ilgili çalışmalar, çoğu için çok önemlidir. Diğer çalışmalar için diyet kısıtlaması başlatmak için sadece bir SDR plaka düzenli bir OP50 plaka solucanlar aktarmak. Bu bölümde, SDR hayvanlar için bir ömür analizi kurmak için bir protokol, bir örnek olarak açıklanmıştır. <ol><li> OP50 birkaç düzenli NGM plakalar üzerine 20-30 üreme aktif yetişkin aktarmak için bir platin solucan toplayıcı kullanın. Deneyler için gerekli yumurta sayısına bağlı olarak, daha fazla yetişkin gerekli olabilir. </li><li> 20 ° C&#39;de 4 saat boyunca yumurtlama izin plakaları bırakın. </li><li> Plaka yetişkin çıkarın. Tabaklar yumurta için görsel olarak kontrol edilmelidir. </li><li> 20 plakaları bırakın ° C ve döl Late-L4/day 1 yetişkin aşamaya geliştirmeye olanak sağlar. Genellikle, bu 20 N2 vahşi türleri solucanlar için 3 gün sürer ° C </li><li> Diyet kısıtlaması başlatmak için uygun bir gün 1 yetişkin SDR tabağa aktarın. </li><li> Ömrü analizi için, plaka başına 10-20 solucanlar, her SDR durum için 6-8 plakalarının toplam kurdu. </li><li> Tüm solucanların ölmüş kadar her 2 günde bir her solucanın canlılığı Puan. Solucanlar gıda yoksunluğu önlemek için her 12-48 saat taze SDR plakaları transfer olması kritik önem taşımaktadır. </li></ol> 4. Temsilcisi Sonuçlar: Şekil 1&#39;de gösterildiği OD 600 ile ilgili OP50 konsantrasyon tahmini için temsili bir kalibrasyon eğrisi bir komplo. Liner regresyon analizi tarafından oluşturulan denklem OP50 bakteri konsantrasyonları gelecekteki tüm hesaplamalar için kullanılabilir. Şekil 2&#39;de gösterildiği gibi farklı solucan suşlarının ortalama ömrü kronik SDR tedavilerin etkisi assaying bir örnektir. Mavi çizgi SDR yabani tip solucanlar normal bir doza bağımlı bir yanıt temsil eder. Daf-16 (mu86) SDR-indüklenen ömrünü uzatma üzerinde hiçbir etkisi sahipken kırmızı çizgi, SDR ömrü etkisi kısmen DRR-2 oe tarafından bastırılmış olduğunu gösterir. C. ömrü analiz gerçekleştirmek için Detay yöntemi elegans önceki edebiyat 1-3 açıklanmıştır. <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="Şekil 1" /> Şekil 1 E. tahmini için bir kalibrasyon eğrisi Temsilcisi sonuç Coli OP50 konsantrasyon. OP50 konsantrasyonu ölçülen OD 600 değerlerinin bir fonksiyonu olarak çizilmiştir. Hata çubukları = 2x, 1.5x, 4x, 8x, 16x, 32x SD Gecelik bakteriyel kültür absorbans ölçümü önce sulandırılmış ve daha seyreltilmiş 1.000 x iki kez kob ölçümden önce oldu. <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="Şekil 2" /> Şekil 2 C Temsilcisi sonucu elegans ömrü deneyi (Ching ve ark Şekil 4) SDR yanıt vahşi tip N2 hayvanlar (mavi) iki farklı mutantlar (yeşil ve kırmızı) ile karşılaştırarak. Hata çubukları = SEM

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Diyet kısıtlaması (DR) ömrünü uzatır ve türlerin 5-7 memeliler dahil olmak üzere geniş bir yelpazede, çeşitli yaşa bağlı hastalıkların başlamasını geciktirmek için bilinir. Hem çevresel ve genetik manipülasyonlar C modeli DR kullanılmıştır elegans kültür solucanlar da dahil olmak üzere tanımlanmış yarı sıvı bir ortamda 8,9, 10-12 sıvı bir ortamda bakteri besin kaynağı seyreltme ve daha yavaş bir hızda 13 yaşında davranış mut...

<table border="1"><tbody><tr><td> Reaktifin Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Carbenicillin </td><td> Duchefa Biochemie </td><td> C0109.25 </td><td> Su içinde çözülür. </td></tr><tr><td> Kanamisin </td><td> Roche </td><td> 10106801001 </td><td> Su içinde çözülür. </td></tr><tr><td> Bacto-pepton </td><td> Becton Dickinson ve şirket </td><td> DF0118072 </td><td></td></tr><tr><td> Bacto-Tripton </td><td> Becton Dickinson ve şirket </td><td> DF0123075 </td><td></td></tr><tr><td> Bacto-maya özü </td><td> Becton Dickinson ve şirket </td><td> DF0127071 </td><td></td></tr><tr><td> Agar </td><td> Becton Dickinson ve şirket </td><td> DF00145170 </td><td></td></tr><tr><td> Steril kültür tüpünün </td><td> ABD Bilimsel </td><td> 1485-0810 </td><td></td></tr><tr><td> Spektrofotometre </td><td> Amersham Biosciences </td><td> Gene Quant pro </td><td> Amersham artık GE Healthcare parçası. </td></tr><tr><td> Diseksiyon Mikroskop </td><td> Zeiss </td><td> STEMI 2000 </td><td></td></tr><tr><td> Santrifüj </td><td> Beckman Coulter </td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

Yetersiz beslenme veya açlık olmadan gıda alımının azaltılması, ömrünü uzatır ve memeliler dahil olmak üzere çeşitli türlerin geniş bir yelpazede, yaşa bağlı hastalıkları, başlangıcından gecikme bilinir. Aynı zamanda vücut ağırlığı ve doğurganlık azalması yanı sıra, plazma glukozu, insülin ve IGF-1, bu hayvanlarda düşük seviyelerde neden olur. Bu tedavi genellikle diyet kısıtlaması (DR) ya da kalori kısıtlaması (CR) olarak adlandırılır. Nematod Caenorhabditis elegans yaşlanma biyoloji eğitimi için önemli bir model organizma olarak ortaya çıkmıştır. Her ikisi de çevresel ve genetik manipülasyonlar modeli DR kullanılmış olan ve C. ömrünü uzatmaya göstermiştir elegans. Ancak, C. içinde bildirilen DR çalışmaları birçok elegans yaşlanma alanında genetik çalışmaların çoğu standart katı agar petri tabak yapıldı, sıvı ortamda hayvanların propaganda yapıldı. Burada öldürülmüş bakteri içeren standart katı NGM agar tabanlı plaka kullanarak DR protokol sunuyoruz.

Watch this Scientific Journal Video about Solid Plate-based Dietary Restriction in Caenorhabditis elegans at JoVE.com

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Burada plaka tabanlı katı diyet kısıtlaması yapmak için bir protokol mevcut<em&gt; C. elegans</em&gt; Öldürülmüş bakteri.

Katı Plaka tabanlı Diyet Kısıtlama Caenorhabditis elegans</em

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

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Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K Görüntüleme.  University of Michigan. Burada plaka tabanlı katı diyet kısıtlaması yapmak için bir protokol mevcut C. elegans Öldürülmüş bakteri.

Video: Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Ölçme Caenorhabditis elegans Katı Medya Ömrü

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

Biyoloji

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

İzolasyon ve In vitro Etkinleştirme Caenorhabditis elegans Sperm

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

Erken Embriyo Oluşumu Zaman atlamalı Mikroskopi Caenorhabditis elegans

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

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

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

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

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

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

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Çoklu doymamış yağ asitlerinin Diyet takviyesi<em&gt; Caenorhabditis elegans</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

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 ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Caenorhabditis eleganlarında Dokuya Özgü Proteostatik Düşüşün Ölçülmesi

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Caenorhabditis elegans Yaş İlişkili Nörodejeneratif Hastalıkların Modellendirilmesi

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

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 ...