This work was supported by Adelphi University faculty development grant and grant R15GM113156 from NIGMS awarded to EVC, grant R01GM067862 from NIGMS and grant R01AG027849 from NIA awarded to DMR.

1. 시약 및 솔루션 <ol><li> 플라이 음식과 미디어의 준비 <ol><li> 열 플라이 재료를 90 ℃로 설정 열판에서 물에 V / W 0.79 % 한천 11 % 설탕, 2 % 자기 분해 된 효모, 5.2 %의 옥수수 가루,. 균질 혼합물을 약간 조밀 얻어 질 때까지 정기적으로 교반한다. 각각의 음식을 비행 5 ㎖와 100 병의 총 비행 음식의 550 ML의 최종 부피를 준비합니다. </li><li> , 열원으로부터 제거 음식 아래 80 ° C로 냉각 될 때까지 때때로 교반하고, 95 % 에탄올에 녹인 0.2 % tegosept 메틸 -4- 히드 록시 벤조 에이트를 추가한다. </li><li> 두 개의 동일한 볼륨에서 음식을 분할합니다. 다른 볼륨에 에탄올 50 mm의 하나의 볼륨에 에탄올 라파 마이신의 1.1 ml의 1.1 ML을 추가합니다. 라파 마이신과 음식에 에탄올 솔루션을 저어. 온도가 약을 추가하기 전에 50 ° C로 감소 있는지 확인하십시오. </li></ol></li><li> 절연 버퍼 <ol><li> 절연 버퍼 [225 mM의 만니톨, 75 mM의 자당, 10 mM의 3- (N &lt;500 ml의 준비/ EM&gt; -morpholino) 프로판 술폰산 (MOPS) 및 1mM의 에틸렌 디아민 테트라 아세트산 (EDTA), 2.5 ㎎ / ㎖ 소 혈청 알부민 (BSA)] 물. </li><li> 수산화 나트륨을 사용하여 7.2로 pH를 조정한다. 초기 pH는 산성이 될 것입니다. </li><li> 용액을 멸균하고 4 ℃에서 보관하는 0.22 ㎛의 공극 크기를 갖는 멸균 필터 저장 병을 사용. </li></ol></li><li> 세척 버퍼 <ol><li> 세척 버퍼 [225 mM의 만니톨, 75 mM의 자당, 10 mM의 KCl을 10 mM 트리스 염산, 5 mM의 KH 2 PO 4] 물 500 mL로 준비합니다. </li><li> 수산화 나트륨을 사용하여 7.2로 pH를 조정한다. 초기 pH는 산성이 될 것입니다. </li><li> 용액을 멸균하고 4 ℃에서 보관하는 0.22 ㎛의 공극 크기를 갖는 멸균 필터 저장 병을 사용. </li></ol></li></ol> 초파리 균주 2. 양육 <ol><li> , 개발 과정에서 유충 밀도를 제어 할 문화 병에 부모 25 쌍을 배치하고 48 시간 동안 알을 낳기 할 수 있도록합니다. </li> &lt;리&gt; 달걀의 밀도를 조절하기 위해 48 시간 후 부모를 제거합니다. </li><li> 반복 신흥 F1 자손을 사용하여 2.1 및 2.2 단계, 그래서이 밀도 제어의 두 세대가 달성된다. </li><li> 성인을 수집, 이산화탄소 (CO 2)와 성을 분리하여 파리 마취. <ol><li> 5 PSI의 연속적인 흐름에 높은 압력을 탱크로부터 전달되는 순수 CO 2 싱글 스테이지 조정기를 사용한다. 정전기를 방지하려면 물에 플라스크를 필터링 500 ml의에 이산화탄소를 촉진하기 위해 플라스틱 튜브를 사용합니다. </li><li> 플라스크를 밀봉 한 구멍이있는 고무 마개를 사용합니다. CO 2 패드에 플라스크의 측면 구멍을 연결하는 플라스틱 튜브를 사용합니다. </li><li> 10 ~ 15 분 이상 더 이상 패드에 파리를 놓습니다. 실험 조건에 배치하기 전에 24 시간 동안 마취에서 복구 할 수 있습니다. </li></ol></li><li> 미토콘드리아 풍부한 분수에 충분한 대사 산물을 생성 샘플 당 300 파리를 사용합니다. 여기에, 여성을 사용하지만, gendeR은 다른 실험에서 다를 수 있습니다. 집에서 만든 1 패 인구 통계학 케이지 (150) 파리를 전송합니다. 유리 병에 플라이 음식을 5 ml에 액세스 할 수 있습니다. </li><li> 또 다른 1 패 케이지에 남아있는 150 파리를 전송합니다. 150 개 이상의 파리와 케이지에 너무 많이하지 마십시오. </li><li> 실험 조건에 따라 여섯 복제 샘플을 사용합니다. 전체 동물 추출물보다 대사 산물을 얻을 수 있기 때문에, 전체 동물의 1 샘플이 분리 생성 (50) 여성 한 케이지 (으)로 운항하는 항공사 전송합니다. 미토콘드리아 분리에 관해서는, 실험 조건에 따라 여섯 복제 샘플을 사용합니다. </li><li> 12 시간 빛과 12 시간의 어둠의주기와 25 ° C에서 장소 케이지. </li><li> 음식 품질을 유지하기 위해 음식의 5 ml의 바이알에서 매 2, 3 일 신선 식품을 제공한다. </li><li> 십일 음식에 파리를 유지한다. 이 단계는 라파 마이신 치료 7 필요합니다. 다른 치료 또는 조건이 다를 수 있습니다. </li></ol> 미토콘드리아 분획 3. 분리 <ol><li> 덤프로 한 케이지 (150 파리)에서 운항하는 항공사유리 테플론 다운스 균질는 냉장 분리 버퍼 1ml를 가득. </li><li> 아래 15 스트로크 유봉 위를 이동하여 균질화. 그대로 미토콘드리아를 유지하기 위해 얼음에 박격포를 유지합니다. </li><li> 4 ℃에서 5 분 300 XG에 1.5 ML 튜브와 원심 분리기로 전송 균질. </li><li> 미토콘드리아에 대해 풍부하게 4 ℃에서 10 분 동안 6,000 XG에 뜨는 원심 분리기를 가져 가라. </li><li> 상층 액은 세포질 부분을 포함 바와 같이, 상층 액을 제거 또는 대체 실험을 위해 사용합니다. 세척 완충액 300 μL에 펠렛을 재현 탁. </li><li> 두 번째 케이지 3.5 - 반복 3.1 단계를 반복합니다. 극저온 microcentrifuge 관에서 모두 케이지의 재현 탁 알약을 결합합니다. </li><li> 4 ℃에서 10 분 동안 6,000 XG에 원심 분리기. </li><li> 뜨는을 취소하고 액체 질소에 펠렛을 플래시 동결. </li><li> -80 ° C 이하의 온도에서 미토콘드리아 풍부한 분수를 저장합니다. <ol><li> 또한, 전체 동물 사전에 대한paration, 극저온 microcentrifuge 관에서 성인을 놓습니다. C 이하의 온도 ° -80에서 액체 질소 저장 성인 병을 플래시 동결. </li></ol></li></ol>

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저자가 공개하는 게 없다.

이 프로토콜에서 가장 중요한 단계는 1) 풍부한 공간에서 충분히 파리를 양육. 그것은 150 개 이상의 파리 각각 인구 통계학 새장에 너무 많이하지 않는 매우 중요하다; 2) 자주 식품 영양 학적 스트레스 및 경쟁을 방지하기 케이지 음식을 변경하는 단계; 3) 4 ° C에서 모든 샘플을 유지하는 것은 미토콘드리아 부분의 분리시 무결성을 보장합니다. 또한 분리 완충액 세척 완충액, 및 사용 전에 유리 - ?...

이 절차의 목적은 초파리를 이용하여 대사 체학 연구에 대한 충분한 미토콘드리아 대사 산물을 산출 풍부한 미토콘드리아 분획을 개발하는 것입니다. 우리의 경험에 의하면, 전체 세포 추출 방법을 사용하여 대사 체학 분석은 초파리의 미묘한 미토콘드리아 대사 산물의 변화를 감지 할 수 없습니다. 그러나, 종래 대사 체학 분석 미토콘드리아 분별은 미토콘드리아 대사 변화를 식별하기 위해 감도를 증가시킨다. 미토콘드리아는 세포가 정상적인 기능 (1)에 필요한 에너지의 90 %를 제공 할 책임이 세포 소기관이다. 최근 몇 년 동안 그것은 미토콘드리아 단지 아데노신 삼인산 (ATP)를 생산하는 것보다 세포 및 유기체 기능에 훨씬 더 역동적 인 역할을하고, 지금 대사 항상성의 2,3의 규제를위한 허브로 인식되는 것을 인식하고있다. 미토콘드리아는 DIS되는 endosymbiotic 프로세스의 결과tinct 미생물 계통 1.5 억년 전에 4 ~ 합병했다. 미토콘드리아에 해당하는 세포 기관으로 발전으로, endosymbiont에서 유전자 신흥 핵 게놈에 통합되었다. 37 유전자가 산화 적 인산화 (5)의 효소 복합체의 서브 유닛이다 미토콘드리아 단백질을 인코딩 13있는 미토콘드리아 DNA에 남아있는 동안 동물 오늘날, 약 1,500 미토콘드리아 단백질은 핵 인코딩됩니다. 미토콘드리아 및 핵 구획 간의 조정은 미토콘드리아 적절한 기능을 유지하는데 필요하다. 여기에 설명 된 방법을 이용하여, 우리는 미토콘드리아 게놈과 핵 사이의 조정 조작의 결과 초파리 미토콘드리아 대사의 변화를 감지 할 수 있었다. 우리는 미토콘드리아 DNA 라 자매 종에서 초파리의 변형을 사용 simulans는 단일 D. 위에 놓는다 핵 배경 6 melanogaster의. 이 &#39;중단&#39;mitonuclear유전자형은 D의 &#39;기본&#39;, 또는 공동 발전 mitonuclear 유전자형을 비교 하였다 네이티브 (D)와 같은 핵 게놈을 들고 melanogaster의 미토콘드리아 DNA를 melanogaster의. D. melanogaster의 및 D simulans mtDNAs 100 ~에 의해 아미노산과 mitonuclear 통신 7,8에 영향을 미칠&gt; 500 동의어로 대체를 다릅니다. 우리는 약리학 적 스트레스에 반응하여 대사 산물의 변화를 연구하기 위해 전체 비행 추출물과 미토콘드리아 농축 추출물을 생성합니다. 여기에서 우리는 미토콘드리아 농축 분수를 사용하는 경우 우리는 (D)를 운반하는 네이티브, 공동 발전 유전자형 사이의 미토콘드리아 대사에 뚜렷한 변화를 감지 것을 보여 melanogaster의의 mtDNAs와 D를 들고 혼란 유전자형 simulans 미토콘드리아 DNA. 반면, 이들 두 유전자형 간의 대사 변화는 전체 플라이 추출물을 이용하는 통상의 방법을 사용하여 미묘. 따라서,이 방법은 mtDNAs이의 미토콘드리아 변화를 중재 방법을 이해하는 경로를 제공다른 약물에 반응.

<table><tbody><tr><td>0.2% tegosept-methyl 4-hydroxybenzoate</td><td>VWR</td><td>AAA14289</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>792799</td><td></td></tr><tr><td>Mannitol</td><td>Sigma-Aldrich</td><td>M4125</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S9378</td><td></td></tr><tr><td>3-(&lt;em&gt;N&lt;/em&gt;-morpholino) propanesulfonic acid (MOPS)</td><td>Sigma-Aldrich</td><td>M1254&amp;nbsp;</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>38057&amp;nbsp;</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>5470</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma-Aldrich</td><td>RES3098T-B7</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>1551139</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pads to anesthetize flies</td><td>Tritech Research</td><td>MINJ-DROS-FP</td><td></td></tr><tr><td>1 L cage</td><td>Web Restaurant Store</td><td>999RD32</td><td></td></tr><tr><td>1 L cage lid</td><td>Web Restaurant Store</td><td>999LRD</td><td></td></tr><tr><td>a glass-teflon dounce homogenizer&amp;nbsp;</td><td>Fisher Scientific</td><td>NC9661231</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>rapamycin&amp;nbsp;</td><td>LC Laboratories&amp;nbsp;</td><td>R-5000</td><td></td></tr><tr><td>anti-porin</td><td>MitoSciences</td><td>MSA03</td><td></td></tr><tr><td>anti-alpha tubulin</td><td>Developmental Studies Hybridoma Bank</td><td>12G10</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>23225</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pad</td><td>Tritech Research, Inc</td><td>MINJ-DROS-FP</td><td></td></tr><tr><td>filter flask</td><td>enasco</td><td>SB08184M</td><td></td></tr><tr><td>rubber stopper</td><td>enasco</td><td>S08512M</td><td></td></tr></tbody></table>

preparation of mitochondrial enriched fractions for metabolic analysis in drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

프로토콜은 위에서 설명 된 사용하여, 우리는 발산 mtDNAs 7 라파 마이신 약물의 효과를 시험하기 위해 미토콘드리아 분획을 농축 및 전체 동물 추출물에 대사 체 분석을 수행 하였다. 우리는 플라이 음식에 약물을 용해하여 라파 마이신의 200 μM을 전달했다. 파리 10 일 라파 마이신에 노출되었다. 전체 비행 추출물로부터 미토콘드리아 대사 추출물로부터 용매 ?...

Watch this Scientific Journal Video about Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila at JoVE.com

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

대사 분석을위한 미토콘드리아 농축 분획의 제조<em&gt; 초파리</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

preparation-mitochondrial-enriched-fractions-for-metabolic-analysis

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

9.5K 조회수.  Adelphi University. Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster. 

비디오: Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

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.

조직의 최소 금액을 사용 하 여 초파리의 Permeabilized 섬유에 미토 콘 드리 아 산소 소비량의 측정

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

연구

생화학

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

초파리 멜라노가스터의 구연산 신타제 활동의 대색 분석

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

가스 크로마토그래피-질량 분석 (GC-MS) 초파리 애벌레 샘플의 준비-대사체학을 기반으로

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

발생학

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

효모 세포 Mitochondria의 정화

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

생물학

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

행동분석학

초파리의 미토콘드리아 생리학 및 생물 에너지학 분석

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

저자 스포트라이트: Drosophila melanogaster를 사용한 미토콘드리아 기능 및 화학적 독성 탐구 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

고분해능 호흡 측정법을 사용한 초파리 멜라노가스터 PINK1B9-Null 돌연변이의 미토콘드리아 기능 분석

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

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.

초파리 melanogaster Larval 및 성인 두뇌의 변화 분석

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

신경과학

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

성적 증명서와 Metabolites에서의 정화<em&gt; Drosophila</em&gt; 헤드

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

대사 분석을위한 미토콘드리아 농축 분획의 제조

요약

더 많은 비디오 탐색

이 비디오의 챕터

조직의 최소 금액을 사용 하 여 초파리의 Permeabilized 섬유에 미토 콘 드리 아 산소 소비량의 측정

초파리 멜라노가스터의 구연산 신타제 활동의 대색 분석

가스 크로마토그래피-질량 분석 (GC-MS) 초파리 애벌레 샘플의 준비-대사체학을 기반으로

효모 세포 Mitochondria의 정화

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

고분해능 호흡 측정법을 사용한 초파리 멜라노가스터 PINK1^B9-Null 돌연변이의 미토콘드리아 기능 분석

고분해능 호흡 측정법을 사용한 초파리 멜라노가스터 PINK1^B9-Null 돌연변이의 미토콘드리아 기능 분석

고분해능 호흡 측정법을 사용한 초파리 멜라노가스터 PINK1^B9-Null 돌연변이의 미토콘드리아 기능 분석

초파리 melanogaster Larval 및 성인 두뇌의 변화 분석

성적 증명서와 Metabolites에서의 정화