وقد تم تطوير هذا البروتوكول من خلال عملنا داخل وبالتعاون مع مختبرات بول مارتن وخاسينتو انطونيو. نشكر مركز ألبوم بلومينغتون لخدمة الممتازة والمجتمع ذبابة الفاكهة لمواصلة تبادل خطوط الطيران. وتمول حاليا من قبل BS منحة المشروع BBSRC. وتمول من قبل WW زمالة يلكوم ترست التطوير الوظيفي.

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+protein+reporters+for+use+with+the+Drosophila+Gal4+expression+system+and+for+vital+detection+of+balancer+chromosomes.">New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes.</a> Genesis. 34, 135-138 (2002).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila protocols. , (2000).</li><li>Tepass, U., Fessler, L. I., Aziz, A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+of+hemocytes+and+their+relationship+to+cell+death+in+Drosophila.">Embryonic origin of hemocytes and their relationship to cell death in Drosophila.</a> Development. 120, 1829-1837 (1994).</li><li>Millard, T. H., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+filopodial+interactions+during+the+zippering+phase+of+Drosophila+dorsal+closure.">Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure.</a> Development. 135, 621-626 (2008).</li><li>Doerflinger, H., Benton, R., Shulman, J. M., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+PAR-1+in+regulating+the+polarised+microtubule+cytoskeleton+in+the+Drosophila+follicular+epithelium.">The role of PAR-1 in regulating the polarised microtubule cytoskeleton in the Drosophila follicular epithelium.</a> Development. 130, 3965-3975 (2003).</li><li>Olofsson, B., Page, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Condensation+of+the+central+nervous+system+in+embryonic+Drosophila+is+inhibited+by+blocking+hemocyte+migration+or+neural+activity.">Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity.</a> Dev Biol. 279, 233-243 (2005).</li><li>Paladi, M., Tepass, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+Rho+GTPases+in+embryonic+blood+cell+migration+in+Drosophila.">Function of Rho GTPases in embryonic blood cell migration in Drosophila.</a> J Cell Sci. 117, 6313-6326 (2004).</li><li>Vlisidou, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+embryos+as+model+systems+for+monitoring+bacterial+infection+in+real+time.">Drosophila embryos as model systems for monitoring bacterial infection in real time.</a> PLoS Pathog. 5, e1000518-e1000518 (2009).</li><li>Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+actin-based+epithelial+adhesion+and+cell+matching+during+Drosophila+dorsal+closure.">Dynamic actin-based epithelial adhesion and cell matching during Drosophila dorsal closure.</a> Curr Biol. 10, 1420-1426 (2000).</li><li>Wood, W., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cell+movement+during+dorsal+closure+in+Drosophila+embryos.">Imaging cell movement during dorsal closure in Drosophila embryos.</a> Methods Mol Biol. 294, 203-210 (2005).</li><li>Kunwar, P. 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وأهم عناصر هذا الإجراء يتم اختيار الأجنة السليمة مع hemocytes المسمى بوضوح ، ويشن منها بعناية من دون تعرضها للتلف. ولا يمكن تصوير الأجنة مرة واحدة في مجال النفط والهالوكربون أنها مقاومة للجفاف وشنت مرة واحدة لعدة ساعات. في أيدينا يمكننا hemocytes صورة لمدة ثلاث ساعات ، مع الجفاف تافهة من الجنين أو واضحة الصور الضرر ، مع الأخذ في Z - كومة من الصور كل ثلاث دقائق على المجهر لدينا LSM510 زايس مبائر مع الهدف 40X. منذ hemocytes ديناميكية للغاية قد يكون هناك مقايضة بين القرار المكانية والزمانية : يجوز إذا كانت هناك حاجة صورة عالية جدا وقرار كرية دموية داخل هياكل تتحرك أثناء الفحص. وسيتم تحديد التفاصيل الدقيقة لكيفية جمع الصور في وقت لاحق من الأسئلة التجريبية لمعالجتها. نستخدم عادة UAS - GFP أو UAS GMA - 3 إلى hemocytes التسمية ؛ UAS - GMA مفيد بشكل خاص منذ ذلك الحين ، بالإضافة إلى وسم hemocytes ، فإنه يوفر للقراءة من ديناميات خيوط الأكتين. ويمكن استخدام بنيات أخرى UAS للتحقيق في السلوك كرية دموية : على سبيل المثال يمكن استغلالها UAS - 9 ، ويبني الكرز fluorophores بديلة أو يمكن استخدامها UAS تاو - 10 - GFP لتصور ميكروتثبول داخل كرية دموية. قد يبني UAS مع تسمية النووية أن تكون ذات قيمة خاصة لتتبع حركات كرية دموية الآلي (الشكل 2E - F). اللونين التصوير الممكن أيضا (على سبيل المثال يمكن لأحد أن تسمية كل من أكتين وcytoskeletons أنيبيب باستخدام UAS - الكرز moesin وUAS - تاو GFP ، على التوالي ، تحت سيطرة سائق Gal4 كرية دموية محددة). كما ذكرنا سابقا ، وأحد المحددات الرئيسية في التصوير المباشر لhemocytes هو السائق Gal4 المستخدمة : فيما يتعلق المروجين لكرية دموية محددة الحالية SRP - Gal4 2&gt; crq Gal4 - 11&gt; pxn Gal4 - 4 ، مع SRP - Gal4 فقط كافية لتسمية hemocytes عندما متغايرة ، في حين أن هناك حاجة إلى ما لا يقل عن نسختين من crq - Gal4 أو pxn Gal4 أن تحتفل hemocytes حية. مع ذلك التصوير الأمثل مع SRP - Gal4 يتطلب جود زيجوت متماثلة الألائل (أو وجود سائق Gal4 البديلة). hemocytes التالية التي تعيش في هذه الطريقة فمن الممكن لدراسة تشتت نموهم من الرأس (8) ونلاحظ كيف تتفاعل مع هذا النمط العظة هذه العملية ، إيداع الجثث المصفوفة وتبتلع أفكارك. Hemocytes تمثل أيضا نظاما للاهتمام الذي لبحث آلية للهجرة الخلية ؛ من خلال دراسة هجرة hemocytes في الأجنة التي تفتقر إلى عناصر cytoskeletons الأكتين أو أنيبيب فمن الممكن أن نفهم وظيفة في الجسم الحي أكثر وضوحا (على سبيل المثال رو GTPases 4 ، 12). استغلال UAS يبني على هذه التسمية بعد cytoskeletons يوفر معلومات أكثر تفصيلا عن تنظيمها. لقد قمنا بتعديل هذه التقنية لتحقيق السلوك كرية دموية في سياقات متنوعة مثل ردودهم على الليزر التي يسببها wounds4 وحقن البكتيريا fluorescently المسمى 13. وعلاوة على ذلك يمكن بسهولة أن تتكيف هذه الطريقة لأنواع الصور خلية أخرى مختلفة عن طريق اختيار Gal4 السائقين. على سبيل المثال قد التقط لنا سابقا إغلاق الظهرية باستخدام السائقين الظهارية Gal4 14 و 15. ويقتصر تطبيق هذه التقنية لأنواع الخلايا الأخرى عن طريق قوة Gal4 السائق ، والموقف من الخلايا المراد تصويره داخل الجنين ونوع الاداة المستخدمة في التصوير ؛ المجاهر multiphoton مبائر تمكين المستخدم من سلوك الخلية صورة عميقة داخل الجنين (مثل الهجرة الخلية الجرثومية بطريق الظهارة 16) ، في حين أن المجاهر مبائر التقليدية غير قادرة على الوصول إلى هذه الأعماق (وهذا هو السبب في أننا الصور على طول خط الوسط حيث بطني hemocytes سطحية جدا ، محاصرين بين الحبل البطني العصب النامية والبشرة). قوة هذا البروتوكول هو أنه يوفر بيئة للحفاظ على الأجنة في حالة صحية جيدة للتصوير حي. في حين أن هناك حاجة إلى درجة من المهارة اليدوية لوضع الأجنة ، فإنه هو أسلوب سهل لإتقان بسرعة ويعطي نتائج يمكن استنساخه وتعديلها بسهولة باستخدام مختلف Gal4 السائقين ويبني UAS للتحقيق في جوانب مختلفة من علم الأحياء كرية دموية. بالإضافة إلى ذلك ، لا يقتصر على بروتوكول hemocytes لأن استخدام برامج بديلة Gal4 تمكن من تحليل السلوك من أنواع الأنسجة الأخرى.

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live imaging of drosophila melanogaster embryonic hemocyte migrations

العديد من الخلايا باستخدام عنوان دراسات الهجرة<em&gt; في المختبر</em&gt; طرق ، في حين أن بيئة ذات الصلة من الناحية الفسيولوجية هي الكائن الحي نفسه. هنا نقدم بروتوكول لمن تصاعد<em&gt; ذبابة الفاكهة السوداء البطن</em&gt; الأجنة الحية والتصوير اللاحقة hemocytes fluorescently المسمى ، والضامة الجنينية من هذا الحي. باستخدام نظام Gal4 - UAS<sup&gt; 1</sup&gt; نحن حملة للتعبير عن مجموعة متنوعة من العلامات ، المرمزة وراثيا في الموسومة fluorescently hemocytes لمتابعة نموهم في جميع أنحاء تفريق الجنين. مجموعة التالية من الأجنة في مرحلة المرجوة من التنمية ، ويتم إزالة المشيماء والخارجي هي التي شنت ثم الأجنة في النفط الهالوكربون بين مسعور ، غشاء الغاز نفاذية وساترة زجاجية للتصوير حي. بالإضافة إلى معلمات الإجمالي المهاجرة مثل السرعة واتجاهها ، وارتفاع القرار التصوير مقرونا استخدام صحفيين الفلورسنت من طراز F - أكتين ميكروتثبول ويمكن أن توفر معلومات أكثر تفصيلا بشأن هيكل الخلية ديناميات هذه المكونات.

Watch this Scientific Journal Video about Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations at JoVE.com

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

hemocytes ذبابة الفاكهة تفريق أكثر من مجمل الجنين النامية. هذا البروتوكول يوضح كيفية تحميل الصور وهذه الهجرات استخدام الاجنة مع hemocytes fluorescently المسمى.

يعيش تصوير ذبابة الفاكهة السوداء البطن هجرات كرية دموية الجنينية

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

live-imaging-of-drosophila-melanogaster-embryonic-hemocyte-migrations

Universidad San Sebastian

Research

JoVE Journal

Biology

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

15.6K Views.  University of Bath. hemocytes ذبابة الفاكهة تفريق أكثر من مجمل الجنين النامية. هذا البروتوكول يوضح كيفية تحميل الصور وهذه الهجرات استخدام الاجنة مع hemocytes fluorescently المسمى.

Video: Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

تشريح الجهاز العصبي المركزي اليرقات في Melanogaster ذبابة الفاكهة

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

يعيش تصوير الدماغ الجنينية اسماك الزرد بواسطة الميكروسكوب متحد البؤر

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

تعيش خلية التصوير والتحليل الكمي من الخلايا الظهارية في الجنينية القيطم المورق</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

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.

تشريح Oenocytes من الكبار ذبابة الفاكهة السوداء البطن</em

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

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

عزلة ذبابة الفاكهة السوداء البطن الخصيات

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

إعداد فردية ذبابة الفاكهة البيض غرف للتصوير لايف

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

يعيش التصوير من البروتينات التي تحمل علامات GFP في<em&gt; ذبابة الفاكهة</em&gt; البويضات

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

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.

قياس عمر في<em&gt; ذبابة الفاكهة الدروسوفيلا</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

يعيش التصوير من تخليص الخلية أفكارك أثناء<em&gt; ذبابة الفاكهة</em&gt; مرحلة التطور الجنيني

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

التصوير من خلال حالة العذراء من<em&gt; ذبابة الفاكهة السوداء البطن</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...