Bu çalışma, NIA YW.CF (AG21068, AG31086) hibe ile desteklendi.

1. İnsülin Çözüm Hazırlığı <ol><li> 0.01 mg / ml konsantrasyonu elde etmek için PBS içinde insülin eriterek taze sığır insülin çözüm hazırlayın. Insülin / PBS ve kontrol PBS iki çözüm de enjeksiyon prosedürü boyunca buz üzerinde tutulmalıdır. Bu çözümler,% 0.5 (v / v) FD &amp; C mavi no ile hazırlanmalıdır. 1 gıda renklendirme. </li></ol> 2. İğne hazırlanması ve Enjeksiyon Set-up <ol><li> Kapiller cam iğneler, çekin, 210; Hız, 100; Zaman, 200 (100ms) Isı, 345: çektirmenin ayarları enjeksiyon için yeterli kalitede iğne üreten aşağıdaki mikropipet puller.The kullanarak hazırlayın. Taze çekilmiş iğne ucu Kimwipe doku üzerinden basarak köreldiğini olmalıdır. Bu küntleşme süreci uzamış yüksek direnç ucu kaldırır ve daha büyük bir gözenek çapı ile stouter ucu üretir. </li><li> Köreldiğini iğne ucu-insülin / yalnız PBS solüsyonu veya PBS içeren bir mikrosantrifüj tüp yerleştirin. Her iğne geri-dolum eylem sonuç Kılcal. Stereomicroscope altında hiçbir enkaz veya hava kabarcıkları ucunda görüntülendiğinden emin olmak için bir iğne ucu uyun. Temiz doldurmak değil herhangi bir iğne atın. </li><li> Iğne ucu bir stereomikroskopta görünür böylece manuel mikroenjektör iğne tutucu ve pozisyon iğne tutucu bir micromanipulator ile doldurulur iğne takın. Mikroenjektör şırınga üzerindeki pimi takılı el mikroenjektör mikrometre düğmesini çevirerek iğne sıvı sütun üzerinde pozitif basınç uygulayın. </li><li> Iğne ucu Kimwipe dokunmadan ve sıvı akışını doğrulayan doğrulayın sıvının yer değiştirmesi için yeterli basınç tatbik edilmiştir. </li><li> İğnenin açık bant yapıştırmayın iğne mili, enjeksiyon için bir kalibrasyon grafiği hazırlanır ve 20X büyütme altında, bir stereomikroskopta iğne ucu ile odak noktası haline getirmek olmuştur. </li></ol> 3. Hazırlık Fly <ol><li> On günlük erkek sineklerin enjeksiyon deneyleri için kullanılmaktadır. Eclosion 24 saat içinde sinekleri toplayın. Erkek yeri ve erkek sineklerin dışarı standart veya tedavisinde diyet içeren flakon içine bir gaz ped nemlendirilmiş CO 2, sıralama sinekler anestezisi. 10 gün boyunca deneysel diyetler erkek sineklerin koruyun. </li><li> Deneysel diyetler eclosion ve ayırma takip eden onuncu gün, transfer, 5 ml% 2 agar fişi içeren şişeleri, gıda içeren flakon uçar ve 12-16 saat açlıktan </li><li> 1 saat süreyle insülin enjeksiyon öncesinde% 10 glukoz batırılmış filtreleri içeren flakon açlıktan sinek aktarın. </li><li> Kısaca glikoz beslenmeden sonra nemlendirilmiş CO 2 sinekler uyutmak ve o zaman soğuk, buz onları hareketsiz. </li></ol> 4. Enjeksiyon Yöntemi <ol><li> Yavaşça ince forseps bir çift ile bir soğuk immobilize sinek kavramak ve ucu sinek toraks sol tarafında ön bölgeye bitişik şekilde iğne ucu yakın tutun. Stereomicroscope altında iğne ucu ve sinek toraks hem de odak noktası haline getirin. </li><li> Iğne ucu tümsek sinek toraks (Şekil 1) sol prescutum bölgenin merkezi değecek şekilde hafifçe sinek iğne ucu doğru hareket ettirin. Sinek düzgün odaklı ve iğne ucu ile uyumlu bir kez, böylece iğne iğne prescutum merkezinde impales üzerine sinek ilerlemeye devam ediyor. </li><li> Sıvı 0.1 ul sinek içine enjekte edilmiş kadar mikroenjektör mikromanipülatör düğmesi ile şırınga pistonu ilerleyen gerektiği gibi pozitif basınç uygulayın. Sinek hemocoel içine sıvı akışı, ön Toraks sol tarafına mavi bir renk verir. Bazen bir geri çekin ve herhangi bir iğne ucu tıkanması yerinden ve sıvı akışı için izin vermek için birkaç kez ilerlemek gerekir. </li><li> Enjekte sinekler hemolimf çıkarma önce belirlenmiş zaman noktaları için% 2 agar şişeleri kurtarmak için izin verin. </li></ol> 5. Hemolimf Koleksiyon <ol><li> Kısaca istenilen enjeksiyon sonrası zaman noktalarında ve pozisyon nemlendirilmiş CO 2 sinekler uyutmak dorsal tarafı aşağı bir CO 2 pad yüzeyine yapıştırılmış çift taraflı bant her sinek. Hizalı kafa kapsül ile satır bile sinek düzenleyin. Bir yapıştırıcı bant kanatları basın ve immobilizasyon sağlamak için kesilmiş ahşap bir aplikatör çubuk kullanabilirsiniz. </li><li> Sinek, bant sabit, ince forseps ile her sineğin burnumun kavramak ve baş kapsül ön stereo mikroskop ile görülebilecek şekilde, hafifçe sinek, karın ve arka sonra bölgeye yönelik çekin. </li><li> Diğer el ile ve burnumun pozisyonda tutarak, delinme ptilinal sütür sadece üzerinde kafa kapsül merkezi keskin bir tungsten iğne kullanılarak (Şekil 2). Baş kapsülü diğer tarafı ile iğne ucu yumruk ve daha sonra c kaybetmek kolaydır Bir çok uzak iğne eklemek için dikkatli olmak gerekirhemolimf akış ontrol. Delinme hemolimf toplama önce bir enjeksiyon grubunda uçar. </li><li> Baş kapsülü delinmiş edildikten sonra, hafifçe sinek karına baskı uygulamak için kesilmiş ahşap bir aplikatör çubuk kullanabilirsiniz. Bu kadar üzerinde baskı uygulamak gibi ventral tarafta aittir sinek rulo için yararlı olabilir. Bu prosedür, baş kapsül ponksiyon sitesinden hemolimf Bir damla üretecek. </li><li> Baş kapsül ponksiyon sitesinde çıkan hemolimf damlacık 1μl microcapillary bir tüp bir ucundan dokunun. Hemolimf kapiller eylem ile tüp girecektir. Mezun bir miktar çizelgesi karşı microcapillary tüp içinde sıvı sütununu kontrol ederek toplanan hemolimf miktarını belirleyin ve kaydedin. </li></ol> 6. Hemolimf Glikoz Belirlenmesi <ol><li> Mikroplaka kuyuları Infinity Glikoz Reaktif 100 ul içeren hemolimf örnekleri çıkarın. Hemolimf örnekleri yüklenirken buz üzerinde plaka tutun. </li><li> 50mm, 25mm, 12.5mm, 6.25mM ve 3.125mM 1 ul şekeri stok çözümlerin her biri ayrı ayrı yükleyerek bir standart eğri oluşturulması. </li><li> 37 numune ° C&#39;de 10 dakika. </li><li> 340 nm&#39;de absorbans algılar. </li><li> Hesap hemolimf hacmi için toplanan ve bilinen glikoz konsantrasyonları ile standart eğri dayalı örnek glukoz konsantrasyonu belirlemek. </li></ol> 7. Temsilcisi Sonuçlar Tipik bir insülin tolerans yanıt glikoz düzeylerinin bir damla enjeksiyon sonrası 15 dakika tespit ve insülin enjekte sinekler tespit edilmiştir. Buna karşılık, bu tür tepki PBS enjekte sinekler (Şekil 3) görülmez. Periferik glukoz bertaraf Bu yanıt insülin enjekte devam enjeksiyon sonrası 30 dakika kadar uçar. Biz, her enjeksiyon grubunda rutin olarak 4-5 sinek başına 0.2-0.5 ul hemolimf ayıklayın. Üç enjeksiyon grubu, her bir deney yer almaktadır. <img src="/files/ftp_upload/2722/2722fig1.jpg" alt="Şekil 1"/> Şekil 1 Drosophila toraks iğne ekleme sitesi (Demerec, 1950 Değiştirilmiş) 7 gösteren sol tarafında. Prescutum, göğüs kafesinin sol tarafında ön, dorsal bölge merkezi aracılığıyla iğne yerleştirin. <img src="/files/ftp_upload/2722/2722fig2.jpg" alt="Şekil 2"/> Şekil 2 hemolimf çıkarımı (Demerec, 1950 Değiştirilmiş) 7 ponksiyon yerini gösteren Drosophila kafa Önden görünüm. Delinme ptilinal sütür sadece üzerinde kafa kapsül merkezinde ince bilenmiş tungsten probu ile baş kapsül. <img src="/files/ftp_upload/2722/2722fig3.jpg" alt="Şekil 3"/> Şekil 3 kontrolü yetişkin tespit tipik bir insülin tolerans yanıt uçar. Kontrol w 1118 sinekler, sığır insülini (PBS içinde 1 ng) veya PBS sadece enjekte edildi . 0, 15 veya 30 dakika ve glikoz düzeyleri dolaşan kurtarmak için çoğaltmak gruplar halinde Sinekler sonra izin verildi ölçüldü.

<ol>
	<li>Rulifson, E. J., Kim, S. K., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+insulin-producing+neurons+in+flies:+growth+and+diabetic+phenotypes.">Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes.</a> Science. 296, 1118-1118 (2002).</li><li>Kim, S. K., Rulifson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+mechanisms+of+glucose+sensing+and+regulation+by+Drosophila+corpora+cardiaca+cells.">Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells.</a> Nature. 431, 316-316 (2004).</li><li>Broughton, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longer+lifespan,+altered+metabolism,+and+stress+resistance+in+Drosophila+from+ablation+of+cells+making+insulin-like+ligands.">Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands.</a> Proc Natl Acad Sci U S A. 102, 3105-3105 (2005).</li><li>Haselton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+ablation+of+adult+Drosophila+insulin-producing+neurons+modulates+glucose+homeostasis+and+extends+life+span+without+insulin+resistance.">Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance.</a> Cell Cycle. 9, 3063-3063 (2010).</li><li>Haselton, A. T., Fridell, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Drosophila+melanogaster+as+a+model+for+the+study+of+glucose+homeostasis.">Adult Drosophila melanogaster as a model for the study of glucose homeostasis.</a> Aging (Albany NY). 2, 523-523 (2010).</li><li>Belgacem, Y. H., Martin, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+insulin+pathways+alters+trehalose+level+and+abolishes+sexual+dimorphism+in+locomotor+activity+in+Drosophila.">Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila.</a> J Neurobiol. 66, 19-19 (2006).</li><li>Demerec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Drosophila. , (1950).</li></ol>

Bu raporda açıklanan teknik Drosophila hemolimf kompozisyon saptanabilir değişiklikler sonucu fizyolojik süreçleri araştıran bir çalışmada, potansiyel olarak yararlı olur. Bu şekilde enjeksiyon ve hemolimf toplama birleştirerek, belirli bir deneysel tedavi veya manipülasyon anında önemli fizyolojik etkilerini tespit etmek mümkündür. Dekapitasyon 2 içeren önceki teknikleri üzerine hemolimf koleksiyonunda bu &quot;katliam&quot; tekniğinin en önemli avantajı, bağırsak content...

<table border="1"><tbody><tr><th> Reaktif / ekipman Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> Sığır insülin </td><td> Sigma </td><td> I5500 </td></tr><tr><td> Infinity Glikoz Reaktif </td><td> Thermo Electron Corporation </td><td> TR1541 </td></tr><tr><td> Manuel mikroenjektör </td><td> Sutter Enstrüman </td><td></td></tr><tr><td> P-87 Flamming / Brown mikropipet çektirmesi </td><td> Sutter Enstrüman </td><td></td></tr><tr><td> Tek varil borosilikat kılcal cam </td><td> AM Sistemleri </td><td> 626000 </td></tr><tr><td> FD &amp; C Blue No: 1 </td><td> McCormick &amp; Company </td><td></td></tr><tr><td> 1 ul microcapillary tüpler </td><td> Drummond </td><td></td></tr><tr><td> Üç eksenli manuel mikromanipülatör ve baz </td><td> Dünya Hassas Aletler </td><td></td></tr></tbody></table>

insulin injection and hemolymph extraction to measure insulin sensitivity in adult drosophila melanogaster

Korunmuş besin algılama mekanizmaları, memeli ve peptidler gelişimsel larva aşamaları 1,2 sırasında glikoz dengesini korumak için sırasıyla memeli insülin ve glukagon, fonksiyon benzeyen meyve sineği arasındaki mevcut . Büyük ölçüde post-mitotik yetişkin sinekleri üzerinde yapılan çalışmalar, insülin-benzeri hücreler (IPC) 3 üreten peptid (ILP) genetik ablasyonu sonucu glikoz dengesini pertürbasyon ortaya koymuştur. Böylece, yetişkin meyve sinekleri, tip II diyabet gibi metabolik bozukluklar için uygun bir genetik model sistem olarak büyük umut tutun. Meyve sineği sistemi daha da geliştirmek için, memelilerde glukoz toleransı ve insülin duyarlılığını ölçmek için kullanılan karşılaştırılabilir fizyolojik testlerin oluşturulması gerekir. Bu amaçla, son zamanlarda erişkin sinek oral glukoz tolerans tepki ölçmek için bir roman prosedürü tarif var ve glikoz dengesini 4,5 korunmasında yetişkin IPCs önemini göstermiştir. Burada insülin enjeksiyonu 6 için daha önce açıklanan prosedür değiştirilmiş ve erişkin sinek periferik insülin duyarlılığını ölçmek için yeni bir hemolimf ekstraksiyon yöntemi ile kombine var. Benzersiz, protokol erişkin sinek insülin enjeksiyonu, insülin sinyal mutantlar ve erişkin sinek glukoz toleransı ve insülin duyarlılığını etkileyen olası müdahaleler karakterize etmek mümkün kılan bir metodoloji üzerine periferik glukoz yükü elden çıkarma yeteneği doğrudan fizyolojik ölçümler sağlar.

Watch this Scientific Journal Video about Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster at JoVE.com

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Korunmuş insülin sinyalizasyonu meyve sineği bulundu yolaklar<em&gt; Drosophila melanogaster</em&gt; Bu organizmanın, tip II diyabet gibi metabolik bozukluklar modelleme için potansiyel bir araç yapmak. Bu amaçla, erişkin sinek periferik glukoz bertaraf edilmesinde etkili sistemik insülin etkisini ölçmek için fizyolojik testleri kurmak için kritik öneme sahiptir.

İnsülin Enjeksiyon ve Yetişkin İnsülin Hassasiyet Tedbir hemolimf Ekstraksiyon Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

insulin-injection-hemolymph-extraction-to-measure-insulin-sensitivity

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Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

19.6K Görüntüleme.  State University of New York. Korunmuş insülin sinyalizasyonu meyve sineği bulundu yolaklar Drosophila melanogaster Bu organizmanın, tip II diyabet gibi metabolik bozukluklar modelleme için potansiyel bir araç yapmak. Bu amaçla, erişkin sinek periferik glukoz bertaraf edilmesinde etkili sistemik insülin etkisini ölçmek için fizyolojik testleri kurmak için kritik öneme sahiptir.

Video: Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Yetişkin Drosophila melanogaster Locomotion Yüksek Çözünürlüklü Video Takip

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Biyoloji

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Yetişkin Zebra balığı retro-orbital Enjeksiyon

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Yetişkin gelen Oenocytes diseksiyonu Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

İnsülin ifade Koloni oluşturan atalarıdır için Kantitatif Testi

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

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 ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

Ramsay Testi Kullanımı sıvı salgılanması ve İyon Flux fiyatlar Tedbir<em&gt; Drosophila melanogaster</em&gt; Malpighi Tübül

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Glikoz Alımı Ölçümü ve İnsülin Uyarıcısına Yanıt<em&gt; In vitro</em&gt; Kültürlü İnsan Primer Miyotları

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Confocal peptit Y-pHluorin, görüntüleme: insülin granül ekzositozu olduğu gibi fare ve insan adacıkları görselleştirmek için bir teknik

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

Drosophila Melanogaster yetişkin dolaylı uçuş kaslarının optik kesitsel kas alanı belirlenmesi

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Bakteriyel enfeksiyon ve atma Drosophila melanogaster içinde sözlü

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

İnsülin Enjeksiyon ve Yetişkin İnsülin Hassasiyet Tedbir hemolimf Ekstraksiyon Drosophila melanogaster

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In ömrü ölçümü

Ramsay Testi Kullanımı sıvı salgılanması ve İyon Flux fiyatlar Tedbir

Glikoz Alımı Ölçümü ve İnsülin Uyarıcısına Yanıt

Confocal peptit Y-pHluorin, görüntüleme: insülin granül ekzositozu olduğu gibi fare ve insan adacıkları görselleştirmek için bir teknik

Drosophila Melanogaster yetişkin dolaylı uçuş kaslarının optik kesitsel kas alanı belirlenmesi

Bakteriyel enfeksiyon ve atma Drosophila melanogaster içinde sözlü