이 작품은 NIA에서 YW.CF (AG21068, AG31086)에 보조금 지원했다.

1. 인슐린 솔루션 준비 <ol><li> 0.01 MG / ML의 농도를 달성하기 위해 PBS에 인슐린을 용해하여 새로운 소 인슐린 솔루션을 준비합니다. 인슐린 / PBS 및 제어 PBS 솔루션 모두 사출 절차를 통해 얼음에 보관해야합니다. 이러한 솔루션은 0.5 % (V / V) FD &amp; C 블루 번호와 함께 준비한다. 1 식용 색소. </li></ol> 2. 니들 준비 및 설정 사출 <ol><li> ; 당겨, 210, 속도, 100, 시간, 200 (100ms) 열, 345 : 풀러 설정이 주사에 대한 충분한 품질의 바늘을 생산 따라 micropipette puller.The를 사용하여 모세관 유리 바늘을 준비합니다. 갓 뽑아 바늘은 Kimwipe 조직을 통해 팁을 눌러 무딘해야합니다. 이 둔화 과정은 길쭉한 높은 저항 팁을 제거하고 큰 기공 직경 stouter 팁을 생산하고 있습니다. </li><li> 팁 - 최대 인슐린 / 혼자 PBS 용액 또는 PBS를 포함하는 microcentrifuge 관에 무딘 바늘을 놓으십시오. 각 바늘의 백업 작성의 작업 결과를 모세. 더 파편이나 공기 방울이 끝에 표시되지 않도록 stereomicroscope 아래 바늘의 팁을 관찰. 정상적으로 입력되지 않은 바늘 폐기하십시오. </li><li> 바늘 끝이 stereomicroscope를 통해 볼 수 있도록 micromanipulator로 수동 microinjector 니들 홀더 및 위치 바늘 홀더에 가득 바​​늘을 삽입합니다. microinjector의 주사기에 플런저에 연결된 수동 microinjector 마이크로 미터 손잡이를 돌려하여 바늘의 유체 열을 긍정적인 압력을 적용합니다. </li><li> 유체 변위 위해 충분한 압력을 확인은 바늘 끝에 Kimwipe를 감동 유체 흐름을 확인하여 적용되었습니다. </li><li> 일단 바늘이 부착 분명 테이프와 바늘 샤프트로, 분사에 대한 보정 차트 준비하고, 20X 배율 아래 stereomicroscope을 통해 초점에 바늘 끝을 가지고있다. </li></ol> 3. 준비 플라이 <ol><li> 텐 일 된 암컷 파리는 분사 실험에 사용됩니다. eclosion 24 H 내에 파리를 수집합니다. 남성 여성과 장소 파리가 밖으로 표준 또는 치료 식단을 포함 유리병에 가스 패드에 humidified CO 2, 정렬과 함께 파리를 마취. 10 일간 실험 다이어트에 대한 여성의 파리를 유지합니다. </li><li> 실험 다이어트에 eclosion 분리 다음 열째 날, 송금은 2 % 한천의 5 ML 플러그인을 포함하는 튜브 자신의 음식을 함유 튜브에서 파리와 12-16 H. 그들을 굶어 </li><li> 인슐린 주입하기 전에 1 시간 10 % 포도당 젖었 필터를 포함하는 튜브에 굶주려 파리를 전송합니다. </li><li> 간단히 포도당 수유 후 humidified CO 2와 함께 파리를 마취 후 차가운 얼음에 그들을 고정. </li></ol> 4. 사출 절차 <ol><li> 부드럽게 잘 포셉 한 쌍의와 추위에 고정 비행을 파악하고 끝은 파리의 흉부의 왼쪽의 앞쪽에 지역에 인접한 있도록 그것은 바늘 끝 가까이에 잡아. stereomicroscope 아래의 초점에 바늘 끝과 파리의 흉부를 모두 가져와. </li><li> 바늘 끝이 파리의 흉부 (그림 1)의 왼쪽 prescutum 지역의 융기의 중심을 접촉 있도록 부드럽게 바늘 끝쪽으로 날아 이동합니다. 파리가 올바르게 지향하고 바늘 끝에 부합되면, 바늘은 prescutum의 중심을 impales 있도록 바늘에 비행을 사전에 계속. </li><li> 유체의 0.1 μl가 비행에 주입되어 때까지 microinjector의 micromanipulator 손잡이와 주사기의 플런저를 발전하여 필요에 따라 긍정적인 압력을 적용합니다. 파리의 hemocoel로 유체의 흐름은 앞쪽에 흉부의 왼쪽에 파란색을 가르친다합니다. 때때로 하나는 취소 및 바늘 팁 차단을 이동시키다 및 유체 흐름 수 있도록 여러 번 사전해야합니다. </li><li> 주입 파리 hemolymph 추출하기 전에 지정된 시간이 지점에 대한 2 % 한천 튜브 안에 복구하도록 허용합니다. </li></ol> 5. Hemolymph 컬렉션 <ol><li> 간단히 원하는 사후 분사 시간 지점과 위치에 humidified CO 2와 함께 파리를 마취 등의 사이드 다운 CO 2 패드의 표면에 부착된 양면 테이프의 각 비행. 정렬 헤드 캡슐도 행으로 파리를 마련. 하나는 테이프 접착제로 자신의 날개를 눌러 고정을 위해 손질 나무 작은 주걱 스틱을 사용할 수 있습니다. </li><li> 일단 파리가 테이프로 고정 좋은 포셉 각 비행의 코을 파악하고 머리 캡슐의 앞에는 스테레오 현미경을 통해 볼 수 있도록 부드럽게 비행의 후부 다음 복부 및 지역으로 그것을 끌어 수 있습니다. </li><li> 다른 손과 위치에 코를​​ 들고 있지만, 펑크 단지 ptilinal 봉합 위의 헤드 캡슐의 중심은 날카로운 텅스텐 바늘 (그림 2)를 사용. 그것이 머리를 캡슐 반대편을 통해 바늘의 팁을 펀치와 이후 C를 잃고 쉽게로서 하나는 너무 멀리 바늘을 삽입하지 않도록주의해야합니다hemolymph 흐름 ontrol. 찔린 모든 hemolymph를 수집하기 전에 사출 그룹에 날아갑니다. </li><li> 머리 캡슐가 구멍되면 부드럽게 파리의 복부에 압력을 적용하는 손질 나무 작은 주걱 스틱을 사용합니다. 그것은 당신이 압력을 적용으로는 복부 측면에 붙어 있도록 동안 파리를 롤 도움이 될 수 있습니다. 이 절차는 머리 캡슐 찔린 사이트에서 hemolymph의 비말을 생산합니다. </li><li> 머리 캡슐 찔린 사이트에서 신흥 hemolymph 비말에 1μl microcapillary 튜브의 한쪽 끝을 터치. hemolymph는 모세관을 통해 튜브를 입력합니다. 졸업 볼륨 차트에 대한 microcapillary 튜브 내의 유체 열을 선택하여 수집한 hemolymph의 양을 확인하고 기록합니다. </li></ol> 6. Hemolymph 포도당 결정 <ol><li> microplate 우물은 무한 포도당 시약 100 μl를 포함하는으로 hemolymph 샘플을 추출. hemolymph 샘플을로드하는 동안 얼음 접시 보관하십시오. </li><li> 50mM, 25mM, 12.5mM, 6.25mM과 3.125mM에서 별도로 1 μl 포도당 주식 솔루션의 각로드하여 표준 곡선을 설정합니다. </li><li> 37 샘플을 품어 ° C를 10 분. </li><li> 340 nm의 흡광도에서 감지. </li><li> hemolymph 볼륨에 대한 계정을 수집하여 알​​려진 포도당 농도와 표준 곡선에 따라 샘플 포도당 농도를 결정한다. </li></ol> 7. 대표 결과 전형적인 인슐린 내성 반응은 포도당 수준을 순환의 드롭 15 분 후 주사를 감지 인슐린 주입 파리에서 감지됩니다. 대조적으로, 이러한 반응은 PBS 주입 파리 (그림 3)을 볼 수 없습니다. 주변 포도당 처리에서이 반응은 인슐린 주입에 계속 삼십분 이후 주사까지 날아. 우리는 정기적으로 각 사출 그룹에 4-5 파리 당 hemolymph의 0.2-0.5 μl 압축을 풉니다. 세 사출 그룹은 각 실험에 포함되어 있습니다. <img src="/files/ftp_upload/2722/2722fig1.jpg" alt="그림 1"/> 그림 1. 주사 바늘을 ​​삽입 사이트 (Demerec 1950에서 수정) 7를 보여주 Drosophila의 흉부의 왼쪽 측면. 흉부의 왼쪽의 앞쪽에, 등의 지역에 prescutum의 중심을 통해 바늘을 삽입합니다. <img src="/files/ftp_upload/2722/2722fig2.jpg" alt="그림 2"/> 그림 2. hemolymph 추출 (Demerec 1950에서 수정) 7 바늘 위치를 보여주는 Drosophila 머리의 전두엽 볼 수 있습니다. 찔린 단지 ptilinal 봉합 위의 헤드 캡슐의 중심에 가늘게 날카롭게 텅스텐 탐침과 함께 머리를 캡슐. <img src="/files/ftp_upload/2722/2722fig3.jpg" alt="그림 3"/> 그림 3. 컨트롤 성인에서 검색된 전형적인 인슐린 내성 응답이 빠르다. 컨트롤 w 1118 파리는 소 인슐린 (PBS 1 NG) 또는 PBS에만 주입했다. 복제 그룹에 날아 그 다음 0, 15 또는 30 분 순환 포도당 수준 복구할 수 있었다 것은 측정했다.

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

관심 없음 충돌 선언하지 않습니다.

이 보고서에 설명된 기술은 Drosophila의 hemolymph 구성에 감지 변경의 결과로 생리 과정을 조사하는 연구에서 잠재적으로 유용합니다. 이러한 방식으로 주입하고 hemolymph 컬렉션을 결합함으로써, 특정 실험 치료 또는 조작의 즉각적인 생리학 관련 효과를 확인할 수 있습니다. 잘린 2와 관련된 이전의 기술을 통해 hemolymph 수집이 &quot;방혈&quot;기술의 주요 장점은 직감 contents.If 치료가 때...

<table border="1"><tbody><tr><th> 시약 / 장비 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 보빈 인슐린 </td><td> 시그마 </td><td> I5500 </td></tr><tr><td> 인피니티 포도당 시약 </td><td> 써모 일렉트론 주식 회사 </td><td> TR1541 </td></tr><tr><td> 수동 microinjector </td><td> 서터 악기 </td><td></td></tr><tr><td> P - 87 Flamming / 브라운 micropipette 풀러 </td><td> 서터 악기 </td><td></td></tr><tr><td> 싱글 배럴 borosilicate 유리 모세관 </td><td> AM 시스템 </td><td> 626000 </td></tr><tr><td> FD &amp; C 블루 제 1 </td><td> 매코믹 &amp; 컴퍼니 </td><td></td></tr><tr><td> 1 μl microcapillary 튜브 </td><td> 드루먼드 </td><td></td></tr><tr><td> 쓰리 축 수동 micromanipulator 및 기지 </td><td> 세계 정밀 계측기 </td><td></td></tr></tbody></table>

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

보존 영양 감지 메커니즘은 펩티드가 발달 애벌레 단계 1,2 중에 포도당 항상성을 유지하기 위해 각각 포유류의 인슐린과 글루카곤, 기능과 유사한 포유류와 과일 날아 사이에 존재합니다. 대부분 사후 mitotic 성인 파리에서 연구가 세포 (IPCs) 3를 생산 인슐린과 같은 펩티드 (ILP)의 유전자 절제의 결과로 포도당 항상성의 섭동을 공개했습니다. 따라서 성인 과일 파리 2 형 당뇨병을 포함하여 신진 대사 장애에 적합한 유전자 모델 시스템으로 큰 약속을 잡아. 추가로 과일 파리 시스템을 개발하려면, 포유류에서 포도당 내성과 인슐린 민감도를 측정하는 데 사용되는 비교 생리학 assays 설립해야합니다. 이를 위해, 우리는 최근 성인 비행에 경구 포도당 내성 응답을 측정하는 소설 절차를 설명하고 포도당 항상성은 4,5 유지 성인 IPCs의 중요성을 보여주었다. 여기, 우리는 인슐린 주사 6 이전에 설명한 절차를 수정하고 성인 비행의 말초 인슐린 민감도를 측정하는 소설 hemolymph 추출 방식으로 결합합니다. 유니 클리 우리 프로토콜은 인슐린 주사, 인슐린 신호 돌연변이 성인 비행에서 포도당 내성과 인슐린 민감도에 영향을 미치는 잠재적인 개입을 특징으로이 가능하게 방법론에 주변 포도당 부하를 처리하는 성인 비행 능력의 직접적인 생리적 측정이 가능합니다.

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

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

보존 인슐린 신호 전달은 과일 파리에서 찾은 경로<em&gt; Drosophila melanogaster</em&gt;이 유기체 유형 II 당뇨병을 포함하여 신진 대사 장애를 모델링에 대한 잠재적인 도구를합니다. 이를 위해서는 효과적으로 성인 비행에서 주변 포도당 처리에 체계적 인슐린 작용을 측정하는 생리 assays를 확립하는 것이 중요합니다.

인슐린 주입 및 성인에서 인슐린 민감도를 측정하는 Hemolymph 추출 Drosophila melanogaster</em

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

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

19.6K 조회수.  State University of New York. 보존 인슐린 신호 전달은 과일 파리에서 찾은 경로 Drosophila melanogaster이 유기체 유형 II 당뇨병을 포함하여 신진 대사 장애를 모델링에 대한 잠재적인 도구를합니다. 이를 위해서는 효과적으로 성인 비행에서 주변 포도당 처리에 체계적 인슐린 작용을 측정하는 생리 assays를 확립하는 것이 중요합니다.

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

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

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

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

성인 Drosophila Melanogaster의 로코의 고해상도 비디오 추적

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

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Dissection of Oenocytes from Adult Drosophila melanogaster

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

Dissection of Oenocytes from Adult Drosophila melanogaster

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

성인에서 Oenocytes의 해부 Drosophila melanogaster</em

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

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

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

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

인슐린을 표현 콜로니를 형성 Progenitors에 대한 정량 분석

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

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

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 ...

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

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

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

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

램지 분석의 사용은 유체 분비 및 이온 플럭스 요금을 측정하는<em&gt; 초파리</em&gt; Malpighian하는 Tubule

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

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

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

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

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

포도당 섭취량 측정 및 인슐린 자극에 대한 반응<em&gt; In Vitro</em&gt; 배양 된 사람의 주요 근육 세포

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

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

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

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

Confocal Neuropeptide Y-pHluorin의 이미징: 그대로 Murine 인간과 독도에 인슐린과 립 Exocytosis를 시각화 하는 기법

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

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

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

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

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

초파리 Melanogaster 성인 간접 비행 근육의 광 단면 근육 면적 결정

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

Oral Bacterial Infection and Shedding in Drosophila melanogaster

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

Oral Bacterial Infection and Shedding in Drosophila melanogaster

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

세균성 감염 및 Shedding 초파리 melanogaster 에

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

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

라이브 드로소필라 멜라노가스터 에그 챔버에서 내인성 mRNA 를 시각화하고 추적

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...