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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>0.2% tegosept -methyl 4-hydroxybenzoate&#160;</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-(N-morpholino) propanesulfonic acid (MOPS)</td>
			<td>Sigma-Aldrich</td>
			<td>M1254&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>38057&#160;</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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T-B7</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>1551139</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 pads to anesthetize flies</td>
			<td>Tritech Research</td>
			<td>MINJ-DROS-FP</td>
			<td></td>
		</tr>
		<tr>
			<td>1 liter cage&#160;</td>
			<td>Web Restaurant Store</td>
			<td>999RD32</td>
			<td></td>
		</tr>
		<tr>
			<td>1 liter cage lid&#160;</td>
			<td>Web Restaurant Store</td>
			<td>999LRD</td>
			<td></td>
		</tr>
		<tr>
			<td>a glass-teflon dounce homogenizer&#160;</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&#160;</td>
			<td>LC Laboratories&#160;</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&#8482; BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 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

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

에반스 블루 염료와 Zebrafish의 애벌레 골격 근육 무결성 분석

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

연구

발생학

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

중간 규모의<em&gt; Drosophila</em&gt; 프로테옴 실험을위한 배아 추출물

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

해 부 및 Immunofluorescent 얼룩의 버섯 몸과 성인 초파리 melanogaster 두뇌 신경 세포 포토 리셉터

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

주변 후 각 조직에 대 한 개발 준비 분자와 초파리 의 Immunohistochemical 분석

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

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 ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Immunohistochemistry, 서쪽 분석 및 RNA 격리에 대 한 초파리 번데기 망막의 해 부

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

초파리 배아에서 근육 수축의 라이브 이미징 및 분석

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

녹고, 경작, 그리고 초파리 셀 라인 Cryopreserving

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

살아있는, 그대로 조직에서 세포 분열의 분석을 위한 초파리 애벌레와 Pupal 고환의 준비

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes&#39; zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve&#39;s regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (&#177; standard deviation) of 137,904 &#177; 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 &#177; 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 &#177; 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P &#60; 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

Here, we present a protocol to significantly reduce the mitochondrial DNA copy numbers in a bovine oocyte (P &lt; 0.0001). This method utilizes centrifugation and bisection to substantially reduce oocyte mitochondria and may allow for an increased chance of development in the reconstructed interspecies somatic cell nuclear transfer embryos.

소 난모세포에서 미토콘드리아 DNA를 감소시키기 위한 Bisection의 사용

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) ...