Name: measurement of metabolic rate in drosophila using respirometry
Uploaded: 2014-06-24T15:00:00
Description: Watch this Scientific Journal Video about Measurement of Metabolic Rate in Drosophila using Respirometry at JoVE.com

우리는 우리의 연구 자금에 대한 최대 플랑크 사회에게 감사의 말씀을 전합니다.

Respirometers 1. 준비 <ol><li> 50 μL 모세관 마이크로 피펫의 삽입을 허용하는 면도날 1,000 μL 피펫 팁을 잘라 가능한 똑바로 피펫 팁을 얻을하려고합니다. </li><li> 피펫으로 거품의 조각을 놓고 피펫 팁에 아래로 누르십시오. </li><li> CO 2 흡수의 작은 금액을 추가하고 폼의 두 번째 부분으로 포함되어 있습니다. </li><li> 마이크로 피펫은 피펫 팁에 삽입되는 장소에 접착제를 적용합니다. </li><li> ?...

<ol>
	<li>Van Voorhies, W. A., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+&amp;#34;rate+of+living&amp;#34;+model:+further+evidence+that+longevity+and+metabolic+rate+are+not+inversely+correlated+in+Drosophila+melanogaster.">Testing the &amp;#34;rate of living&amp;#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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-specific+decrease+in+aerobic+efficiency+associated+with+increase+in+oxygen+free+radical+production+in+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+contribution:+long-lived+Drosophila+melanogaster.+lines+exhibit+normal+metabolic+rates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+modulates+metabolic+rate+and+temperature+sensitivity+in+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+temperature+preferences+and+energy+homeostasis+in+dystroglycan+mutants.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+muscular+dystrophy:+a+genetic+screen+for+dystroglycan+and+dystrophin+interactors+in+Drosophila.+identifies+cellular+stress+response+components.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthermic+seizures+and+aberrant+cellular+homeostasis+in+Drosophila+dystrophic.+muscles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+and+metabolism+in+Drosophila+melanogaster:+genetic+correlations+between+life+span+and+age-specific+metabolic+rate+in+populations+artificially+selected+for+long+life.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+equivalents+of+CO2+and+their+importance+in+assessing+energy+expenditure+when+using+tracer+techniques.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epicurean+fly:+using+Drosophila+melanogaster.+to+study+metabolism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+flies+and+men:+insights+on+organismal+metabolism+from+fruit+flies.">Of flies and men: insights on organismal metabolism from fruit flies.</a> BMC Biology. 11, 38 (2013).</li></ol>

이 프로토콜에서는, 우리는 파리에있는 CO 2 생산을 측정하기위한 저렴하고 신뢰할 수있는 방법에 대해 설명합니다. 우리는이 실험을 수행하는 빠르고, 쉽고, 다른 연구 1, 6, 9와 계약에 재생 가능한 데이터를 생성하는 것으로 나타났습니다. 여기에 설명 된 프로토콜은 쉽게 연구소의 예산 및 재료에 맞게 수정할 수 있습니다. 각 개별 호흡계의 구조는 한 챔버가 밀폐 남아 적응 될 ...

생화학 Kreb의주기는 탄수화물, 지방, 그리고 CO 2 생성 단백질에서 파생 된 아세트산의 산화를 통해 ATP를 생성합니다. 초파리에서, O 2 입력은 직접 CO 2 출력과의 상관 관계 및 대사 1의 수준을 반영한다. 따라서, CO 2 출력의 측정은 성공적 노화와 신진 대사 2-5 관련 연구에 이용되고있다. 여기에 우리의 실험실은 전문 장비가 필요없이 최대 18 개의 시료에서 CO 2 생산을 측정 할 수 있도록, 이전에 설계 실험 설정을 수정했다. 기타 우리는 이전에 근육 영양 장애 관련 단백질, Dystroglycan (DG) 6-8 불충분 파리의 신진 대사 속도의 차이를 보여주기 위해이 방법을 사용했습니다. O 산화 대사에 사용되는 2 호흡 폐기물로 배출되는 CO 2로 변환됩니다. construc손으로 만든 respirometers의 기는 O 2 소모의 속도의 측정을 위해 허용하는 기술되어있다. 파리 효율적으로 기체 상태에서 제거, CO 2 배출을 흡수하는 물질로 밀폐 용기에 배치됩니다. 가스 부피 (감소 압력)의 변화는 폐쇄 호흡계에 부착 유리 모세관 유체의 변위에 의해 측정된다. 다른 것보다이 기술의 주요 장점은 비용이다. 이전 연구는 가스 분석기 및 기술적으로 진보 된 respirometry 시스템 1.9을 사용하여 초파리에 의한 CO 2 생산을 측정했다. 더 복잡한 장비에도 불구하고, 여기에 기재된 방법의 감도는보고 된 값 (표 1)과 유사하다. 또한, 여러 가지 다른 그룹은 초파리 4-6에서 상대 신진 대사 속도를 결정하기 위해이 기술의 변화를 사용했습니다. 따라서, 이러한 분석은 reliab에를 생성하는데 사용될 수있다르, 모든 실험실에 설치 될 수 있으며, 교육 목적으로 사용할 수있는 특수 장비를 구입하지 않고 초파리의 신진 대사에 관련된 데이터를 재현. 일반적으로, 생체의 신진 대사를 결정하는 허용 기법 CO 2 생산을 측정하는 소비 O 2 또는 둘 3,4,9. 하지만, 그것은 O 2의 1 당량은 CO 2의 1 당량을 생성하는 가정 할 수있다, CO 2의 정확한 비율은 발생 10을 활용 한 대사 기판에 따라 달라집니다. 따라서 정확하게 에너지 단위 대사량을 결정하기 위하여 그것 O이 소비 및 CO 2 생산 양을 측정 할 필요가있다. 이 때문에, 여기에 기재된 방법은 동물이 아닌 절대 값 사이의 CO 2 생산의 차이를 비교하는 특별히 중요한 것이다. 우리의 기술은 포의 기간에 걸쳐 여러 동물 CO 2 생산을 통합따라서 전자 (1 ~ 2 시간) 및 동물의 활동의 평균을 반환합니다. 실험 동물은 측정이 다른 활동의 수준과 반드시​​ 신진 대사를 반영 할 수있는 제어 동물보다 활성화되어 있다고 믿을만한 이유가있는 경우.

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			<td height="21" style="height:21px;width:401px;">BlauBrand IntraMark 50&#181;l micropipettes</td>
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			<td style="width:119px;">612-1413</td>
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			<td height="21" style="height:21px;width:401px;">Soda Lime</td>
			<td style="width:101px;">Wako&#160;</td>
			<td style="width:119px;">CDN6847</td>
			<td></td>
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			<td height="21" style="height:21px;width:401px;">Eosine&#160;</td>
			<td style="width:101px;">Sigma&#160;</td>
			<td style="width:119px;">031M4359</td>
			<td>Any dye that can create visible colorization of liquid can be used</td>
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			<td height="22" style="height:22px;width:401px;">Thin Layer Chromatorgaphy (TLC) Developing Chamber</td>
			<td style="width:101px;">VWR</td>
			<td>21432-761</td>
			<td>Any transparent glass chamber that can be closed with the lid</td>
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			<td height="21" style="height:21px;">Anesthetizer, Lull-A-Fly Kit</td>
			<td style="width:101px;">Flinn</td>
			<td>FB1438</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Power Gel Glue</td>
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			<td height="21" style="height:21px;width:401px;">1 ml pipett tips</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Foam&#160;</td>
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			<td></td>
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			<td height="21" style="height:21px;">Plaesticine Putty</td>
			<td style="width:101px;">Any</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:401px;">Scalpel</td>
			<td style="width:101px;">Any</td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:401px;">Twezzers&#160;</td>
			<td style="width:101px;">Any</td>
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			<td></td>
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measurement of metabolic rate in drosophila using respirometry

대사 장애는 인간의 건강에 영향을 미치는 문제가 자주 발생합니다. 따라서 신진 대사를 조절하는 메커니즘을 이해하는 것은 매우 중요한 과학적 작업입니다. 인간의 많은 질병의 원인이 유전자는 초파리의 다른 질환의 개발에 관련된 신호 전달 경로를 연구 할 수있는 좋은 모델을 만들고, 비행 동성이있다. 또한, 초파리의 온순함은 신진 대사를 조절 할 수 있습니다 새로운 치료 표적을 식별에 도움이 유전 화면을 단순화합니다. 이러한 화면을 수행하기 위해 파리의 대사 상태의 변화를 확인하는 간단하고 빠른 방법이 필요하다. 일반적으로, 이산화탄소 생산 산화 기질 및 에너지 비용이 대사 상태에 대한 정보를 제공하는 좋은 지표이다. 이 프로토콜에서 우리는 파리에서 CO 2의 출력을 측정 할 수있는 간단한 방법을 소개합니다. 이 기술은 잠재적으로 신진 대사 속도에 영향을 미치는 유전 적 교란의 식별에 도움이 될 수 있습니다.

이 방법은 우리가 야생형 (오레곤 R)에서 CO 2 생산 남성은 18, 25 일에 파리, 29 ° C 및 DG에 돌연변이 파리를 측정 민감하다는 것을 보여주기 위해. 파리는 25 ° C에서 제기 한 후 측정에 앞서 5 일 동안 실험 온도로 이동했다. 이 ectothermic 종 예상 한 바와 같이, CO 2의 양은 온도 (그림 2)로 증가 생산. 우리는 과거에 설탕 무료 다이어트는 모두 야생 유형의 신진 ...

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

대사 질환은 인간에서 가장 흔한 질병 중 하나들이다. 유 전적으로 다루기 쉬운 모델 생물 D. melanogaster의 대사를 조절하는 신규 한 유전자를 확인하는데 사용될 수있다. 이 논문은 자신의 CO 2 생산을 측정하여 파리의 신진 대사 속도를 연구 할 수있는 비교적 간단한 방법을 설명합니다.

대사율의 측정에<em&gt; 초파리</em&gt; Respirometry를 사용하여

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

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JoVE Journal

Biology

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.

Drosophila의 유충에서 냄새 기반 행동의 고해상도 측정

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

연구

생물학

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.

의 수명 측정<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.

의식 쥐의 사구체 여과율의 측정을위한 높은 처리량 방법

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.

비행 성능의 정확하고 빠른 측정을위한 개선 된 방법에<em&gt; 초파리</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.

에서 애벌레 활동의 측정<em&gt; 초파리</em&gt; 활동 모니터

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.

세포 외 산 생산의 측정 및 분석은 당분 속도를 확인하는

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

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human disease in this organism. Sensitive methods for measurements of parameters such as metabolic rates are needed to drive the understanding of physiology and disease in small animals such as the fruit fly. The method described here assesses fuel oxidation in small numbers of adult flies fed food containing trace amounts of 14C-labeled substrates such as glucose or fatty acid. After the feeding period and any additional experimental manipulations, flies are transferred to short tubes capped with mesh, which are then placed in glass vials containing KOH-saturated filter paper that traps exhaled, radiolabeled CO2 generated from oxidation of radiolabeled substrates as potassium bicarbonate, KHCO3. This radiolabeled bicarbonate is measured by scintillation counting. This is a quantitative, reproducible, and simple approach for the study of fuel oxidation. The use of radiolabeled glucose, fatty acids, or amino acids allows determination of the contribution of these different fuel sources to energy metabolism under different conditions such as feeding and fasting and in different genetic backgrounds. This complements other approaches used to measure in vivo energy metabolism and should further the understanding of metabolic regulation.

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

This paper describes a method for the measurement of fuel oxidation in Drosophila melanogaster in which trace amounts of specific radiolabeled metabolic substrates are fed to flies. The exhaled radiolabeled CO2 that is a produced from fuel oxidation is collected and measured.

방사성 표지 된 기판에서 이산화탄소 생산에서의 측정<em&gt; 노랑 초파리</em

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

장 내분비 세포의 굿 호르몬 분비의 연구 및 라이브 세포 이미징을위한 의도 된 쥐 소장의 혼합 차 문화

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Explanted 망막 조직 세포 외 유출 분석을 사용 하 여 신진 대사 에너지의 측정

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.