Biz araştırma için fon Max-Planck Toplum teşekkür etmek istiyorum.

Respirometers 1. Hazırlanması <ol><li> 50 ul kılcal mikropipet sokulmasına izin verecek şekilde bir jilet ile 1000 ul pipet Kes, mümkün olduğunca düz pipet almaya çalışın. </li><li> Pipet içine köpük bir parça koyun ve pipet aşağı itin. </li><li> CO emici 2 küçük bir miktar ilave edin ve bir köpük ikinci bir parça içermesi. </li><li> Mikropipet pipet ucu sokulur yerde yapıştırıcı uygulanır. </li><li> Tutkal kuruması için bir gecede respirometredeki bırakın. Bir respirometredeki şematik Şekil 1A&#39;da gösterilmiştir. </li></ol> Ölçüm Odası 2. Hazırlanması <ol><li> Görünür bir renklendirme neden olur bir oranda eosin ile su karıştırılarak oda çözelti hazırlayın. </li><li> Bölmenin içine eosin / su çözeltisi dökün. </li><li> Bir santimetre ölçekli bölmenin iki biri etiketleyin. </li></ol><ol><li> Bir işaretleyici ile bireysel respirometers etiketleyin. </li><li> 2 CO ve her respirometredeki içindeki istenen genotip 3-5 sinekleri yerleştirmek için alternatif bir yöntem kullanarak sinek anestezisi. </li><li> Mumlu macunu kullanmanızı sıkıca üstündeki respirometers mühür. </li><li> Sinekler, yaklaşık 15 dakika boyunca anesthetization kurtarmak için izin verin. </li><li> Atmosferik kontrol olarak kullanılacak olan, sinekler olmadan tek solunumu hazırlayın. </li></ol> 4.. Experiment Sahne <ol><li> Odasının üstündeki üst ve alt açık olan bir 1.5 ml Eppendorf tüp tutucu takılarak odası içindeki respirometers asın. </li><li> Aşağı ucu renkli bir çözelti içine daldırın izin bölmenin içine mikro pipet ile respirometers yerleştirin. </li><li> Sıcaklık ve basınç fluct daha güçlü izolasyon sağlamak için kapak örtüsü ve odası arasında vazelin eklekarışma-sının önlenmesidir. </li><li> Kapağı kapatın ve sistem 15 dakika boyunca dengelenmeye bırakın. </li><li> Her mikropipet içerisindeki sıvının seviyesi görünür olduğundan emin odasının bir fotoğraf çekmek ve böylece ölçek (Şekil 1B gösterildiği örneğe bakınız). </li><li> 1-2 saat sonra, başka resim çekmek. </li><li> Deney bittiğinde, respirometers gelen sinekler kaldırmak ve istenirse tartmak ya da daha fazla gerekirse flakon onları geri aktarın. </li></ol> Sonuçlar 5. Analizi <ol><li> Açık ImageJ yazılımı 11 kullanarak görüntüleri satın aldı. </li><li> Her resimde ölçeği kullanılarak, yazılım piksel ölçekleme ayarlayın. </li><li> Sıvı başında (d1) alınan görüntülerde belirlenen referans noktaya gitti ki mesafeyi (Δd) ve deney sonu (d2) ölçün. Şematik bir örnek, Şekil 1C &#39;de gösterilmiştir. </li><li> Formül üretilen CO2 (ul / saat / sinek) miktarını hesaplayın: </li></ol><img alt="Şekil 1" fo:content-width="2in" src="/files/ftp_upload/51681/51681eq1.jpg" /> Santimetre mikropipet borunun yarıçapı R = Δd = mesafe sıvı santimetre ölçülen test numunelerinin mikropipet kadar taşındı Δc = mesafe sıvı (sinekler olmayan) negatif kontrol numunesinin Mikropipet içine taşındı kullanılan sinek n = h = saat

<ol><li>Van Voorhies, W. A., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Testing+the+%26%2334%3Brate+of+living%26%2334%3B+model%3A+further+evidence+that+longevity+and+metabolic+rate+are+not+inversely+correlated+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Testing the &#34;rate of living&#34; model: further evidence that longevity and metabolic rate are not inversely correlated in Drosophila melanogaster.</a> J Appl Physiol. 97, 1915-1922 (2004).</li>
<li>Ross, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-specific+decrease+in+aerobic+efficiency+associated+with+increase+in+oxygen+free+radical+production+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Journal+of+Insect+Physiology%22%5BJournal%5D)">Age-specific decrease in aerobic efficiency associated with increase in oxygen free radical production in Drosophila melanogaster.</a> Journal of Insect Physiology. 46, 1477-1480 (2000).</li>
<li>van Voorhies, W. A., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selected+contribution%3A+long-lived+Drosophila+melanogaster.+lines+exhibit+normal+metabolic+rates%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Selected contribution: long-lived Drosophila melanogaster. lines exhibit normal metabolic rates.</a> J Appl Physiol. 95, 2605-2613 (2003).</li>
<li>Hulbert, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+rate+is+not+reduced+by+dietary-restriction+or+by+lowered+insulin%2FIGF-1+signalling+and+is+not+correlated+with+individual+lifespan+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Experimental+Gerontology%22%5BJournal%5D)">Metabolic rate is not reduced by dietary-restriction or by lowered insulin/IGF-1 signalling and is not correlated with individual lifespan in Drosophila melanogaster.</a> Experimental Gerontology. 39, 1137-1143 (2004).</li>
<li>Ueno, T., Tomita, J., Kume, S., Kume, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopamine+modulates+metabolic+rate+and+temperature+sensitivity+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Dopamine modulates metabolic rate and temperature sensitivity in Drosophila melanogaster.</a> PLoS ONE. 7, (2012).</li>
<li>Takeuchi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+temperature+preferences+and+energy+homeostasis+in+dystroglycan+mutants%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Changes in temperature preferences and energy homeostasis in dystroglycan mutants.</a> Science. 323, 1740-1743 (2009).</li>
<li>Kucherenko, M. M., Marrone, A. K., Rishko, V. M., Magliarelli Hde, F., Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stress+and+muscular+dystrophy%3A+a+genetic+screen+for+dystroglycan+and+dystrophin+interactors+in+Drosophila.+identifies+cellular+stress+response+components%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Stress and muscular dystrophy: a genetic screen for dystroglycan and dystrophin interactors in Drosophila. identifies cellular stress response components.</a> Developmental Biology. 352, 228-242 (2011).</li>
<li>Marrone, A. K., Kucherenko, M. M., Wiek, R., Gopfert, M. C., Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperthermic+seizures+and+aberrant+cellular+homeostasis+in+Drosophila+dystrophic.+muscles%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Hyperthermic seizures and aberrant cellular homeostasis in Drosophila dystrophic. muscles.</a> Scientific Reports. 1, 47 (2011).</li>
<li>Khazaeli, A. A., Van Voorhies, W., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Longevity+and+metabolism+in+Drosophila+melanogaster%3A+genetic+correlations+between+life+span+and+age-specific+metabolic+rate+in+populations+artificially+selected+for+long+life%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Longevity and metabolism in Drosophila melanogaster: genetic correlations between life span and age-specific metabolic rate in populations artificially selected for long life.</a> Genetics. 169, 231-242 (2005).</li>
<li>Elia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy+equivalents+of+CO2+and+their+importance+in+assessing+energy+expenditure+when+using+tracer+techniques%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">Energy equivalents of CO2 and their importance in assessing energy expenditure when using tracer techniques.</a> The American Journal of Physiology. 260, (1991).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li>
<li>Bharucha, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+epicurean+fly%3A+using+Drosophila+melanogaster.+to+study+metabolism%5BTitle%5D)+AND+%22Pediatric+Research%22%5BJournal%5D)">The epicurean fly: using Drosophila melanogaster. to study metabolism.</a> Pediatric Research. 65, 132-137 (2009).</li>
<li>Rajan, A., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Of+flies+and+men%3A+insights+on+organismal+metabolism+from+fruit+flies%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">Of flies and men: insights on organismal metabolism from fruit flies.</a> BMC Biology. 11, 38 (2013).</li>
</ol>

Biz ifşa hiçbir şey yok.

Bu protokol, biz sinekler CO 2 üretimini ölçmek için ucuz ve güvenilir bir yöntem açıklanmaktadır. Biz bu deney yapmak için hızlı, kolay ve diğer çalışmalar 1, 6, 9 ile uyum içinde olan tekrarlanabilir verilerini oluşturur bulundu. Burada özetlenen protokol kolayca herhangi bir laboratuvarın bütçe ve mevcut materyalleri uyacak şekilde modifiye edilebilir. Her bir respirometredeki inşaat sürece odacık hava geçirmez olarak kalır uyarlanabilir. Ancak, daha uzun, daha ince m...

Biyokimyasal Krebs döngüsü karbonhidratlar, yağlar, ve CO2 üretimi proteinlerden türetilen asetat oksidasyonu ile ATP üretir. Drosophila, O 2 giriş, doğrudan CO 2 çıkışı ile ilişkilidir ve metabolizma 1 düzeyini yansıtır. Bu durumda, CO2 çıkışının ölçümü başarılı yaşlanma ve metabolizma 2-5 ile ilgili çalışmalarda kullanılmıştır. İşte laboratuar herhangi bir özel ekipman gerektirmeden kadar onsekiz örneklerinde CO 2 üretimi ölçümü sağlayan, önceden tasarlanmış deney düzeneklerini değiştirdi. Diğerleri ve daha önce müsküler distrofi ilişkili protein, distroglikan (DG) 6-8 eksiktir sinekler metabolik oranlarındaki farklılıkları göstermek için bu yöntemi kullandık. O oksidatif metabolizma için kullanılan 2 solunum atık olarak atılır CO 2, dönüştürülür. Inşaatel yapımı respirometers tion of O 2 tüketilen oranının belirlenmesi için izin veren tarif edilmektedir. Sinekler verimli gaz fazdan ortadan kaldırarak, CO2 absorbe sürülen bir madde ile kapalı bir kap içine yerleştirilir. Gaz hacmi (azalan basınç) içindeki değişiklik kapalı respirometredeki bağlı bir cam kılcal sıvı değiştirmesi ile ölçülür. Diğerleri üzerinde bu tekniğin önemli avantajı maliyetidir. Önceki çalışmalar gaz analizörleri ve teknik olarak gelişmiş Respirometrik sistemlerini 1,9 kullanarak Drosophila CO 2 üretimini ölçtük. Daha karmaşık ekipman rağmen, burada tarif edilen yöntemin duyarlılığı rapor edilen değerlere (Tablo 1) benzer. Ayrıca, çeşitli gruplar Drosophila 4-6 göreceli metabolik oranlarını belirlemek için bu tekniğin varyasyonları kullandık. Bu nedenle, bu deney, reliab oluşturmak için kullanılabilirle, herhangi bir laboratuarda kurulum olabilir ve eğitim amaçlı kullanılabilecek özel ekipman alımı olmadan Drosophila metabolizması ile ilgili tekrarlanabilir veri. Genel olarak, bir organizmanın metabolizmasını belirlemek için kabul edilen teknikler CO2 üretilen ölçmek için, tüketilen O 2, 3,4,9 ya da her ikisi. Olsa da, O 2 bir eşdeğer CO 2 bir eşdeğer oluşturur varsayılabilir, CO 2&#39;nin tam oranı, üretilen 10, kullanılan alt-tabaka metabolik bağlıdır. Bu nedenle, doğru bir enerji birimi metabolik oranını belirlemek it O 2 tüketilen ve CO2 üretilen iki ölçmek gereklidir. Bu nedenle, burada tarif edilen yöntem, hayvanlara olup mutlak değeri arasındaki CO2 üretimi farklılıkların karşılaştırılması için özel olarak uygundur. Bizim teknik tim bir süre içinde birden fazla hayvan CO2 üretimi entegreBu nedenle e (1-2 saat) ve hayvanların aktivitesinin bir ortalamasını verir. Deney hayvanları ölçümü farklı aktivite düzeyleri ve ille metabolizmasını yansıtan olabilir kontrol hayvanlara göre daha az aktif olduğuna inanmak için bir neden varsa.

<table><tbody><tr><td>BlauBrand IntraMark 50 &amp;micro;l micropipettes</td><td>VWR</td><td>612-1413</td><td></td></tr><tr><td>Soda Lime</td><td>Wako</td><td>CDN6847</td><td></td></tr><tr><td>Eosine&amp;nbsp;</td><td>Sigma</td><td>031M4359</td><td>Any dye that can create visible colorization of liquid can be used</td></tr><tr><td>Thin Layer Chromatorgaphy (TLC) Developing Chamber</td><td>VWR</td><td>21432-761</td><td>Any transparent glass chamber that can be closed with the lid</td></tr><tr><td>Anesthetizer, Lull-A-Fly Kit</td><td>Flinn</td><td>FB1438</td><td></td></tr><tr><td>Power Gel Glue</td><td>Pritt</td><td></td><td></td></tr><tr><td>1 ml pipett tips</td><td>Any</td><td></td><td></td></tr><tr><td>Foam</td><td>Any</td><td></td><td></td></tr><tr><td>Plaesticine Putty</td><td>Any</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Any</td><td></td><td></td></tr><tr><td>Tweezers</td><td>Any</td><td></td><td></td></tr></tbody></table>

measurement of metabolic rate in drosophila using respirometry

Metabolik bozukluklar, insan sağlığını etkileyen sık görülen bir problemdir. Bu nedenle, metabolizmayı düzenleyen mekanizmaları anlamak çok önemli bilimsel bir iştir. İnsanlarda birçok hastalığa neden olan genler Drosophila farklı hastalıkların gelişiminde rol sinyal yolları incelemek için iyi bir model yapma, bir sinek homologunu var. Buna ek olarak, Drosophila tractability metabolizmasını düzenleyen yönelik yeni terapötik hedeflerin belirlenmesine yardımcı olması için genetik ekranlar kolaylaştırır. Bu türdeki bir süzgeçle gerçekleştirmek için sinekler metabolik durum değişiklikleri belirlemek için basit ve hızlı bir yöntem gereklidir. Genel olarak, karbon dioksit üretim substrat oksidasyon ve enerji harcaması, metabolik durumu hakkında bilgi veren iyi bir göstergedir. Bu protokolde sinekler CO 2 çıkışını ölçmek için basit bir yöntem tanıtmak. Bu teknik potansiyel metabolik hızını etkileyen genetik pertürbasyonların belirlenmesinde yardımcı olabilir.

Yöntem, yaban tip (Oregon R) CO 2 üretimini erkek, 18, ​​25 uçar ve 29 ° C ve Dg için mutantını uçan ölçülen duyarlı olduğunu göstermek için. Sinekler 25 ° C yükseltilmiş ve daha sonra ölçümden önce 5 gün boyunca deney sıcaklığına kadar kaydırılmıştır. Bu ectothermic türler için beklendiği gibi, CO2 miktarı sıcaklığında (Şekil 2) ile artış üretti. Biz geçmişte şekersiz diyet hem vahşi tip metabolik hızı azaltı...

Watch this Scientific Journal Video about Measurement of Metabolic Rate in Drosophila using Respirometry at JoVE.com

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolik bozukluklar, insanlarda en yaygın hastalıklardan biri arasında yer alıyor. Genetik uysal bir model organizma D. melanogaster metabolizmasını düzenleyen yeni genleri tanımlamak için kullanılabilir. Bu çalışma da CO 2 üretimini ölçerek sinekler metabolizma hızı okuyan sağlayan nispeten basit bir yöntem tarif etmektedir.

Metabolik Oranı Ölçümü<em&gt; Drosophila</em&gt; Respirometrik kullanarak

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

measurement-of-metabolic-rate-in-drosophila-using-respirometry

Research

JoVE Journal

Biology

Measurement of Metabolic Rate in Drosophila using Respirometry

19.6K Görüntüleme.  Max Planck Institute for Biophysical Chemistry.  Metabolik bozukluklar, insanlarda en yaygın hastalıklardan biri arasında yer alıyor. Genetik uysal bir model organizma D. melanogaster metabolizmasını düzenleyen yeni genleri tanımlamak için kullanılabilir. Bu çalışma da CO 2 üretimini ölçerek sinekler metabolizma hızı okuyan sağlayan nispeten basit bir yöntem tarif etmektedir. 

Video: Measurement of Metabolic Rate in Drosophila using Respirometry

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Yüksek çözünürlüklü Ölçüm Drosophila Larva Koku-Driven Davranış

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Biyoloji

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

Doku çok az miktarda kullanarak Drosophila Permeabilized liflerinin mitokondrial oksijen harcama ölçme noktalar

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Biyokimya

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Drosophila melanogaster Larval ve yetişkin beyin metabolik Analizi

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Nörobilim

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 ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Bilinçli Farelerde Glomerüler Filtrasyon Hızı Ölçümü için bir Yüksek verimli Yöntemi

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Uçuş Performans Doğru ve hızlı ölçüm için geliştirilmiş Yöntemi<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Larvaların Faaliyet Ölçümü<em&gt; Drosophila</em&gt; Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Drosophila için Mikrorespirometrinin Uyarlanması - CASK Mutant Metabolizmasına İlişkin Görüşler

Measuring O2 Consumption in Drosophila melanogaster Using Coulometric Microrespirometry

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable environment. A coulometric microrespirometer consists of an airtight chamber in which O2 is consumed and the CO2 produced by the organism is removed by an absorbent medium. The resulting pressure decrease triggers electrolytic O2 production, and the amount of O2 produced is measured by recording the amount of charge used to generate it. In the present study, the method has been adapted to Drosophila melanogaster tested in small groups, with the sensitivity of the apparatus and the environmental conditions optimized for high stability. The amount of O2 consumed by wildtype flies in this apparatus is consistent with that measured by previous studies. Mass-specific O2 consumption by CASK mutants, which are smaller and known to be less active, was not different from congenic controls. However, the small size of CASK mutants resulted in a significant reduction in O2 consumption on a per-fly basis. Therefore, the microrespirometer is capable of measuring O2 consumption in D. melanogaster, can distinguish modest differences between genotypes, and adds a versatile tool for measuring metabolic rates.

Yazar Spot Işığı: Sıcaklığın Drosophila melanogaster ve Çöle Adapte Böcekler Üzerindeki Etkisi 

Coulometric respirometry is ideal for measuring the metabolic rate of small organisms. When adapted for Drosophila melanogaster in the present study, measured O2 consumption was within the range reported for wildtype D. melanogaster by previous studies. Per-fly O2 consumption by CASK mutants, which are smaller and less active, was significantly lower than the wildtype.

Kulometrik Mikrorespirometri Kullanılarak Drosophila melanogaster'de O2 Tüketiminin Ölçülmesi

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable ...

Metabolik Oranı Ölçümü

Özet

Daha Fazla Video Keşfet

Yüksek çözünürlüklü Ölçüm Drosophila Larva Koku-Driven Davranış

Doku çok az miktarda kullanarak Drosophila Permeabilized liflerinin mitokondrial oksijen harcama ölçme noktalar

Drosophila melanogaster Larval ve yetişkin beyin metabolik Analizi

In ömrü ölçümü

Bilinçli Farelerde Glomerüler Filtrasyon Hızı Ölçümü için bir Yüksek verimli Yöntemi

Uçuş Performans Doğru ve hızlı ölçüm için geliştirilmiş Yöntemi

Larvaların Faaliyet Ölçümü

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

Kulometrik Mikrorespirometri Kullanılarak Drosophila melanogaster'de _O2 Tüketiminin Ölçülmesi

Kulometrik Mikrorespirometri Kullanılarak Drosophila melanogaster'de _O2 Tüketiminin Ölçülmesi