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. الكواشف وحلول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد الطعام ذبابة وسائل الإعلام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حرارة المكونات الذبابة 11٪ سكر، 2٪ الخميرة autolyzed، 5.2٪ دقيق الذرة، أجار 0.79٪ ث / ت في الماء في موقد وضعت في 90 ° C. اثارة بانتظام حتى يتم الحصول على خليط متجانس كثيف قليلا. إعداد الحجم النهائي من 550 مل من ذبابة الغذائية لما مجموعه 100 قارورة مع 5 مل يطير الطعام في كل منهما. </li><li style=";text-align:right;direction:rtl"> إزالة من مصدر التدفئة، وإثارة أحيانا حتى الطعام يبرد إلى 80 درجة مئوية وإضافة 0.2٪ tegosept ميثيل 4-hydroxybenzoate الذائبة في الايثانول 95٪. </li><li style=";text-align:right;direction:rtl"> تقسيم الطعام في مجلدين على قدم المساواة. إضافة 1.1 مل من 50 ملي rapamycin يذوب في الإيثانول إلى مجلد واحد و 1.1 مل من الايثانول لحجم الآخر. تحريك rapamycin والحلول الإيثانول إلى الطعام. مما لا شك فيه أن درجة الحرارة تنخفض إلى 50 درجة مئوية قبل إضافة المخدرات. </li></ol></li><li style=";text-align:right;direction:rtl"> العزلة العازلة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 500 مل من العزلة العازلة [225 ملي المانيتول، 75 ملي السكروز، 10 مم 3- (N &lt;/ م&gt; -morpholino) حمض propanesulfonic (اجتماعات الأطراف) و1 ملم ثنائي أمين الإيثيلين رباعي حمض الخل (EDTA)، و 2.5 ملغ / مل ألبومين المصل البقري (BSA)] في الماء. </li><li style=";text-align:right;direction:rtl"> ضبط درجة الحموضة إلى 7.2 باستخدام هيدروكسيد الصوديوم. إن الأس الهيدروجيني الأولية يكون الحمضية. </li><li style=";text-align:right;direction:rtl"> استخدام معقم زجاجات تخزين التصفية مع حجم المسام 0.22 ميكرون لتعقيم الحل وتخزينها في 4 درجات مئوية. </li></ol></li><li style=";text-align:right;direction:rtl"> غسل العازلة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 500 مل من غسل العازلة [225 ملي المانيتول، 75 ملي السكروز، 10 ملي بوكل، 10 ملي تريس حمض الهيدروكلوريك و 5 ملي KH 2 PO 4] في الماء. </li><li style=";text-align:right;direction:rtl"> ضبط درجة الحموضة إلى 7.2 باستخدام هيدروكسيد الصوديوم. إن الأس الهيدروجيني الأولية يكون الحمضية. </li><li style=";text-align:right;direction:rtl"> استخدام معقم زجاجات تخزين التصفية مع حجم المسام 0.22 ميكرون لتعقيم الحل وتخزينها في 4 درجات مئوية. </li></ol></li></ol> 2. تربية من سلالات ذبابة الفاكهة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> للسيطرة على كثافة اليرقات خلال التنمية، وضع 25 زوجا من الآباء والأمهات في لزجاجة الثقافة والسماح لهم لوضع البيض لمدة 48 ساعة. </li> &lt;لى&gt; إزالة أولياء الأمور بعد 48 ساعة لتنظيم كثافة البيض. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 2.1 و 2.2 باستخدام ذرية F1 الناشئة، حتى يتم تحقيق جيلين من هذه السيطرة الكثافة. </li><li style=";text-align:right;direction:rtl"> لجمع الكبار، تخدير الذباب باستخدام ثاني أكسيد الكربون (CO 2) ومنفصلة حسب الجنس. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام مرحلة منظم واحد لتسليم CO النقي 2 من خزان ضغوط عالية في تدفق مستمر من 5 رطل. لتجنب الكهرباء الساكنة، واستخدام أنابيب بلاستيكية لتحفيز CO 2 إلى 500 مل تصفية قارورة بالماء. </li><li style=";text-align:right;direction:rtl"> استخدام سدادة مطاطية مع فتحة واحدة لاغلاق القارورة. استخدام أنابيب بلاستيكية لتوصيل الفتحة الجانبية للقارورة لوحة CO 2. </li><li style=";text-align:right;direction:rtl"> وضع الذباب في لوحة لمدة لا تتجاوز 10-15 دقيقة. السماح للتعافي من التخدير لمدة 24 ساعة قبل وضعها في ظروف تجريبية. </li></ol></li><li style=";text-align:right;direction:rtl"> لتوليد ما يكفي من نواتج الأيض لكسور المخصب الميتوكوندريا، استخدم 300 الذباب في العينة. هنا، استخدام الإناث، ولكن gendeص قد تختلف في تجارب أخرى. نقل 150 الذباب إلى محلية الصنع 1 L الديموغرافيا القفص. السماح بالوصول إلى 5 مل من ذبابة الطعام في قارورة. </li><li style=";text-align:right;direction:rtl"> نقل 150 الذباب المتبقية إلى 1 L قفص آخر. لا overpopulate أقفاص مع أكثر من 150 الذباب. </li><li style=";text-align:right;direction:rtl"> استخدام ست عينات تكرار في حالة تجريبية. منذ مقتطفات الحيوانية كلها تسفر عن مزيد من نواتج الأيض، لتوليد العزلات 1 عينة من الحيوان كله، ونقل 50 أنثى الذباب في قفص واحد. أما بالنسبة للعزلة الميتوكوندريا، استخدم ست عينات تكرار في حالة تجريبية. </li><li style=";text-align:right;direction:rtl"> مكان أقفاص عند 25 درجة مئوية مع دورة 12 ساعة ضوء و 12 ساعة الظلام. </li><li style=";text-align:right;direction:rtl"> توفير المواد الغذائية الطازجة كل 2 أو 3 أيام في قارورة مع 5 مل من المواد الغذائية للمحافظة على جودة الطعام. </li><li style=";text-align:right;direction:rtl"> الحفاظ على الذباب على الطعام لمدة 10 يوما. هناك حاجة إلى هذه الخطوة لعلاج rapamycin 7. والعلاج أو غيره من الظروف تختلف. </li></ol> 3. عزل الميتوكوندريا الكسور <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تفريغ الذباب من قفص واحد (150 الذباب) إلىالزجاج تفلون dounce الخالط مليئة 1 مل من العازلة عزل المبردة. </li><li style=";text-align:right;direction:rtl"> التجانس من خلال نقل ما يصل مدقة وهبوطا لمدة 15 السكتات الدماغية. إبقاء هاون على الجليد للحفاظ على الميتوكوندريا سليمة. </li><li style=";text-align:right;direction:rtl"> نقل جناسة لأنبوب 1.5 مل وأجهزة الطرد المركزي في 300 x ج لمدة 5 دقائق في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> اتخاذ طاف والطرد المركزي في 6000 x ج لمدة 10 دقيقة على 4 درجات مئوية إلى إثراء للالميتوكوندريا. </li><li style=";text-align:right;direction:rtl"> كما يحتوي على طاف الكسر عصاري خلوي، تجاهل طاف أو استخدامه لاجراء تجارب بديلة. Resuspend وبيليه في 300 ميكرولتر من غسل العازلة. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 3،1-3،5 لالقفص الثاني. الجمع بين الكريات معلق على حد سواء أقفاص في أنبوب microcentrifuge المبردة. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي في 6000 x ج لمدة 10 دقيقة على 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> تجاهل طاف وفلاش تجميد بيليه في النيتروجين السائل. </li><li style=";text-align:right;direction:rtl"> تخزين الكسور المخصب الميتوكوندريا في -80 ° C أو انخفاض درجة الحرارة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بدلا من ذلك، لمرحلة ما قبل الحيوان كلهparation، ضع البالغين في أنبوب microcentrifuge المبردة. فلاش تجميد القارورة في النيتروجين السائل ومخزن البالغين في -80 ° C أو انخفاض درجة الحرارة. </li></ol></li></ol>

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الكتاب ليس لديهم ما يكشف.

الخطوات الأكثر أهمية في هذا البروتوكول هي: 1) تربية كافية الذباب في الفضاء وفيرة. من المهم جدا أن لا overpopulate الأقفاص الديموغرافيا مع أكثر من 150 الذباب لكل منهما؛ 2) تغيير الغذائي للأقفاص في كثير من الأحيان لتجنب المنافسة المواد الغذائية والنقص الغذائي. و3) الحفاظ على جمي?...

الهدف من هذا الإجراء هو تطوير الكسور الميتوكوندريا المخصب التي تحقق ما يكفي من نواتج الأيض الميتوكوندريا لالايض الدراسات باستخدام ذبابة الفاكهة السوداء البطن. في تجربتنا، والايض التحليل باستخدام أساليب الاستخراج الخلوية كلها غير قادرة على اكتشاف التغيرات الطفيفة الأيض الميتوكوندريا في ذبابة الفاكهة. ومع ذلك، تجزئة الميتوكوندريا قبل التحليل الايض يزيد من حساسية لتحديد التحولات الأيض الميتوكوندريا. الميتوكوندريا هي العضيات الخلوية مسؤولة عن توفير 90٪ من الطاقة التي الخلايا بحاجة لوظيفة عادية 1. في السنوات الأخيرة تم الاعتراف بأن الميتوكوندريا تلعب دورا أكثر نشاطا في وظيفة الخلوية والعضوي من مجرد إنتاج أدينوسين ثلاثي الفوسفات (ATP)، ويتم الاعتراف الآن كمراكز لتنظيم التمثيل الغذائي التوازن 2،3. الميتوكوندريا هي نتيجة لعملية endosymbiotic التي ديساندمجت الأنساب الميكروبية صبغة ~ 1.5 مليار سنة مضت 4. كما الميتوكوندريا تطورت إلى العضيات الحقيقية، وأدرجت الجينات من معايش جواني في الجينوم النووي الناشئة. في الحيوانات اليوم، ما يقرب من 1500 بروتينات الميتوكوندريا هي المشفرة النووية، بينما لا تزال 37 الجينات في الحمض النووي المتقدري، 13 منها ترميز البروتينات الميتوكوندريا التي هي مفارز من المجمعات انزيم الفسفرة التأكسدية 5. هناك حاجة إلى التنسيق بين الميتوكوندريا ومقصورات النووية للحفاظ على وظيفة الميتوكوندريا سليمة. باستخدام الأساليب المذكورة هنا كنا قادرين على اكتشاف التغيرات الأيضية الميتوكوندريا في ذبابة الفاكهة التي تنتج عن التلاعب في التنسيق بين جينوم الميتوكوندريا والنووية. كنا سلالة من ذبابة الفاكهة التي و mtDNA من الأنواع الشقيقة D. وضعت simulans على D. واحد البطن خلفية النووية 6. هذا mitonuclear &#39;عطلت&#39;ومقارنة التركيب الوراثي إلى التركيب الوراثي mitonuclear &quot;الأصلي&quot;، أو تطورت المشارك لD. البطن تحمل نفس الجينوم النووي مع D. شعبه الأصيل البطن و mtDNA. وD. البطن وD. simulans mtDNAs تختلف باختلاف ~ 100 الأحماض الأمينية و&gt; 500 بدائل مرادفة التي تؤثر mitonuclear 7،8 الاتصالات. ولدت لنا مقتطفات ذبابة كاملة ومقتطفات المخصب الميتوكوندريا لدراسة التحولات الأيض ردا على الإجهاد الدوائي. نحن هنا تظهر أنه عند استخدام الميتوكوندريا المخصب الكسور نكتشف التحولات وضوحا في الأيض الميتوكوندريا بين الأصلي،-تطورت شارك الوراثي تحمل D. mtDNAs البطن وراثى تعطلت تحمل D. simulans و mtDNA. في المقابل، فإن التغييرات الأيض بين هذه الأنماط الجينية هما خفية باستخدام أساليب العادية التي تستخدم كله استخراج الطاير. ولذلك، قدمت هذه الطريقة مسار لفهم كيفية mtDNAs توسط التغييرات في الميتوكوندريااستجابة إلى أدوية مختلفة.

<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.

عن طريق شرح بروتوكول أعلاه، أجرينا تحليل metabolomic على الكسور المخصب الميتوكوندريا ومقتطفات الحيوانية كلها لاختبار تأثير المخدرات rapamycin على mtDNAs المتباينة 7. لقد نجينا 200 ميكرومتر من rapamycin عن طريق إذابة الدواء في الغذاء الطاير. تعرضت الذباب إلى ...

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.

إعداد الميتوكوندريا المخصب الكسور لتحليل التمثيل الغذائي في<em&gt; ذبابة الفاكهة</em

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

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JoVE Journal

Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

9.5K Views.  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. 

Video: 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.

تحليل الزرد اليرقات الهيكل العظمي النزاهة العضلات مع ايفانز الأزرق صبغ

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

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).

إعداد على نطاق متوسط ​​من<em&gt; ذبابة الفاكهة</em&gt; مقتطفات الجنين للتجارب بروتيوميك

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.

تشريح وتلطيخ إيممونوفلوريسسينت للفطر الجسم وعلم الخلايا العصبية في أدمغة البالغين melanogaster المورفولوجية

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.

إعداد وضع الأنسجة حاسة الشم الطرفية الجزيئية والتحليل المناعي في المورفولوجية

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.

إعداد عينات اليرقات المورفولوجية للفصل اللوني للغاز-الكتلي (GC-MS)-على أساس جميع

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;

تشريح الشبكية الخوادر المورفولوجية إيمونوهيستوتشيميستري والتحليل الغربي، وعزل الحمض النووي الريبي

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.

التصوير الحي وتحليل تقلصات العضلات في جنين دروسوفيلا

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.

ذوبان الجليد واستزراع كريوبريسيرفينج خطوط الخلايا المورفولوجية

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.

إعداد دروسوفيلا رفال واختبارات Pupal لتحليل انقسام الخلايا في الأنسجة الحية والسليمة

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.

استخدام التقسيم الثنائي لتقليل الحمض النووي للميتوكوندريا في بويضة البقر

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