وبفضل الأعضاء في مرفق مطيافية الكتلة جامعة إنديانا وجامعة يوتا جميع مرفق الأساسية للمساعدة في الاستفادة المثلى من هذا البروتوكول. J.M.T. معتمد من قبل المعهد الوطني للعلوم الطبية العامة "معاهد الصحة الوطنية في" إطار جائزة رقم R35GM119557.

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الكتاب ليس لها علاقة بالكشف عن.

جميع يوفر فرصة لا مثيل لها لدراسة التفاعلات الأيضية التي تؤلف أيض وسيطة. الحساسية لهذه التكنولوجيا، ومع ذلك، يجعل البيانات عرضه للخلفية الوراثية والعظة التنموية، ومجموعة متنوعة من الضغوط البيئية، بما في ذلك درجة الحرارة، والرطوبة، والكثافة السكانية، وتوافر المغذيات. ولذلك، عالية يتطلب...

ذبابة الفاكهة melanogaster المورفولوجية برز كنظام مثالي لدراسة الآلية الجزيئية التي تنظم الأيض الوسيطة. ليس فقط هي حفظت معظم الأيضية بين البشر و المورفولوجية ، ولكن أجهزة الاستشعار المغذيات الرئيسية ومنظمات النمو، مثل الأنسولين، تور، وحركة، تنشط أيضا في أن يطير1،2. كنتيجة لذلك، يمكن استخدام المورفولوجية لاستكشاف الأساس الأيضي للأمراض البشرية تتراوح بين مرض السكري والسمنة نيوروديجينيريشن والسرطان. وفي هذا الصدد، وضع اليرقات المورفولوجية يوفر الإطار المثالي لدراسة برنامج الاستقلابية المعروفة بتحلل الهوائية، أو تأثير واربورغ. تماما كما استخدم العديد من الأورام تحلل الهوائية لتوليد طاقة الكتلة الأحيائية من الكربوهيدرات، حتى للقيام المورفولوجية اليرقات تنشيط تحلل الهوائية لتعزيز النمو التنموي3،،من45. أوجه التشابه هذه بين اليرقات والتمثيل الغذائي الورم تنشئ المورفولوجية كنموذج رئيسي لفهم كيف الهوائية تحلل التنظيم في فيفو.
وعلى الرغم من حقيقة أن الطاير قد برز كنموذج شعبية لدراسة الأيض، تعتمد معظم الدراسات المورفولوجية على الأساليب التي تم تصميمها لقياس نواتج الأيض الفردية3، مثل تريهالوسي أو الشحوم ATP. منذ بروتوكول معين مطلوب لقياس كل المستقلب، الدراسات على أساس مقايسة كثيفة العمالة ومكلفة، ومنحازة إلى تلك المركبات التي يمكن أن تقاس باستخدام مجموعات تجارية. التوصل إلى حل لهذه القيود برز من الحقل لجميع، الذي يوفر وسيلة أكثر كفاءة وغير متحيزة لدراسة الأيض المورفولوجية . خلافا لدراسة على أساس المقايسة، يمكن إجراء تحليل metabolomic واحد في نفس الوقت قياس المئات من جزيء صغير من الأيض وتوفير فهم شامل للكائن الحي حالة ايضية6،7. هذا الأسلوب اتسع إلى حد كبير نطاق الدراسات المورفولوجية الأيضية ويمثل مستقبل هذا المجال الناشئ8.
وتجري دراسات Metabolomic أساسا باستخدام ثلاث تقنيات: (ط) الرنين المغناطيسي النووي (الرنين المغناطيسي النووي) و (ثانيا) السائل اللوني-الكتلي (LC-MS) (ثالثا) الفصل اللوني للغاز-قياس الطيف الكتلي (GC-MS)9. يقدم كل نهج متميز من مزايا وعيوب، وكل من هذه التقنيات قد استخدمت بنجاح دراسة الأيض المورفولوجية . نظراً للبحث الذي أجرى في المختبر لدينا يركز على نواتج الأيض الصغيرة، والقطبية، نحن نوظف أسلوب GC-MS-تعتمد أساسا. GC-MS ويوفر للمستخدم مع عدد من المزايا، بما في ذلك إمكانية تكرار نتائج عالية، القرار الذروة، والحساسية، وتوفر مكتبة الطيفية الأثر (الصناعات الاستخراجية) إلكترون القياسية التي تسمح التعرف السريع على اكتشاف الأيضية ميزات10،11. ومع ذلك، إعداد العينات ل GC-MS، معقد نوعا ما ويتطلب عناية بالتفاصيل. يجب جمع العينات وغسلها ووزنه والمجمدة بطريقة يروي بسرعة التفاعلات الأيضية. وعلاوة على ذلك، الذبيحة يطير مقاوم للبروتوكولات القياسية التجانس ويتطلب طاحونة حبة لضمان استخراج المستقلب الأمثل. وأخيراً، يجب أن تخضع العينات التي تم تحليلها بواسطة GC-MS derivatization الكيميائية قبل الكشف عن12. بينما تصف الأساليب المنشورة سابقا كل هذه الخطوات3،،من1314، يزال يلزم بروتوكول بصرية التي تسمح للمستخدم المبتدئ بتكاثر توليد بيانات عالية الجودة. هنا نظهر كيفية تحضير عينات اليرقات المورفولوجية لتحليل جميع GC-MS-تعتمد. يسمح هذا البروتوكول المستخدم لقياس العديد من نواتج الأيض القطبية الصغيرة التي تؤلف استقلاب الكربون وسط تكاثر.

<table border="0" cellpadding="0" cellspacing="0" style="width:827px;" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Unsulfured blackstrap molasses</td>
			<td style="width:141px;">Good Food, INC</td>
			<td style="width:141px;"></td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila Agar Type II</td>
			<td>Genesee Scientific</td>
			<td>66-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyridine</td>
			<td>EMD Millipore</td>
			<td>PX2012-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methoxyamine hydrocholoride (MOX)</td>
			<td>MP Biomedicals, LLC</td>
			<td>155405</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MSTFA with 1% trimethylchlorosilane</td>
			<td>Sigma</td>
			<td>69478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fleischmann&#8217;s Active dry yeast</td>
			<td>AB Mauri Food Inc</td>
			<td>2192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6oz Drosophila stock bottle</td>
			<td>Genesee Scientific</td>
			<td>32-130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soft tissue homogenizing mix (2 mL tubes)&#160;</td>
			<td>Omni International</td>
			<td>SKU:19-627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vial insert, 250 &#181;L deactivated glass with polymer feet</td>
			<td>Agilent</td>
			<td>5181-8872</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Succinic acid-2,2,3,3-d4</td>
			<td>Sigma</td>
			<td>293075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SpeedVac</td>
			<td>Thermo&#160;</td>
			<td>SC210A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">o-Phosphoric acid</td>
			<td>Fisher Scientific</td>
			<td>A242-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propionic acid</td>
			<td>Sigma</td>
			<td>P5561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-Hydroxy benzoic acid methyl ester</td>
			<td>Genesee Scientific</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bead Ruptor</td>
			<td>Omni International</td>
			<td>SKU:19-040E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ThermoMixer F1.5</td>
			<td>Eppendorf</td>
			<td>5384000012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MultiTherm Shaker with a 24 X 12 mm block</td>
			<td>Benchmark Scientific</td>
			<td>H5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sigma</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL centrifuge tube</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 35 X 10 mm tissue culture dish</td>
			<td>Corning Incorporated</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GC column</td>
			<td>Phenomex</td>
			<td>ZB-5MSi</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of drosophila larval samples for gas chromatography-mass spectrometry (gc-ms)-based metabolomics

وقد أنشأت التطورات الأخيرة في هذا مجال جميع ذبابة الفاكهة melanogaster المورفولوجية كنموذج وراثية قوية لدراسة الأيض الحيواني. عن طريق الجمع بين مجموعة واسعة من أدوات المورفولوجية الوراثية مع القدرة على مسح مساحات كبيرة من الأيض الوسيطة، يمكن أن تكشف عن نهج جميع التفاعلات المعقدة بين النظام الغذائي والوراثي وتاريخ حياة الأحداث ومنبهات بيئية. وبالإضافة إلى ذلك، يمكن اكتشاف الآليات الانزيمية رواية جميع الدراسات وكشف اتصالات مجهولة بين المسارات الأيضية التي تبدو متباينة. من أجل تيسير توسيع نطاق استخدام هذه التكنولوجيا مجتمع المورفولوجية ، هنا نقدم بروتوكول تفصيلية التي توضح هذه المقالة كيفية إعداد عينات اليرقات المورفولوجية للفصل اللوني للغاز-الكتلي (GC-MS)- على أساس تحليل metabolomic. لدينا بروتوكول يتضمن وصفاً لجمع العينات اليرقات واستخراج المستقلب، derivatization الكيميائية وتحليل GC-MS. النجاح في إنجاز هذا البروتوكول سوف تسمح للمستخدمين لقياس الوفرة النسبية لنواتج الأيض القطبية الصغيرة، بما في ذلك الأحماض الأمينية والسكريات والأحماض العضوية تشارك في دورات TCA وتحلل.

لاكتات نازعة المسوخ (دلده)، والتي تفتقر إلى النشاط دلده4، وعناصر مطابقة وراثيا وجمعت يرقات منتصف L2 وتجهيزها وفقا للبروتوكول المذكورة أعلاه. عند مقارنة مع عناصر التحكم، اليرقات متحولة يحمل تغييرات كبيرة في لاكتات، بيروفات ول-2-هيدروكسيجلوتاراتي<sup class="xre...

Watch this Scientific Journal Video about Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics at JoVE.com

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

ويصف هذا البروتوكول كيفية إعداد اليرقات المورفولوجية لتحليل GC-MS-تعتمد metabolomic.

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 ...

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Developmental Biology

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

11.0K Views.  Indiana University. ويصف هذا البروتوكول كيفية إعداد اليرقات المورفولوجية لتحليل GC-MS-تعتمد metabolomic.

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

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.

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 ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

تشريح اليرقات الزرد الغدد التناسلية الأنسجة

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

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, ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

إنشاء الزرد اليرقات "نموذج الحيوان" للتحقيق مثقبيه الكروزية حركية في فيفو

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

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 ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

فحص لسلامة حاجز الدم في الدماغ في دروسوفيلا melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

تحديد حجم الكبد في لارفال زيبرافيش باستخدام المجهر برايتفيلد

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

تطوير نموذج عدوى حمار وحشي لـ Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

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 ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

تشريح وكيمياء الهيستولوجية المناعية وتركيب أدمغة ذبابة الفاكهة اليرقية والبالغة لتصور الفص البصري

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

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

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Chapters in this video

إعداد الميتوكوندريا المخصب الكسور لتحليل التمثيل الغذائي في

تشريح اليرقات الزرد الغدد التناسلية الأنسجة

إعداد على نطاق متوسط من

إنشاء الزرد اليرقات "نموذج الحيوان" للتحقيق مثقبيه الكروزية حركية في فيفو

ذوبان الجليد واستزراع كريوبريسيرفينج خطوط الخلايا المورفولوجية

فحص لسلامة حاجز الدم في الدماغ في دروسوفيلا melanogaster

تحديد حجم الكبد في لارفال زيبرافيش باستخدام المجهر برايتفيلد

تطوير نموذج عدوى حمار وحشي لـ Clostridioides difficile

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

تشريح وكيمياء الهيستولوجية المناعية وتركيب أدمغة ذبابة الفاكهة اليرقية والبالغة لتصور الفص البصري

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

Summary

Explore More Videos

Chapters in this video

إعداد الميتوكوندريا المخصب الكسور لتحليل التمثيل الغذائي في

تشريح اليرقات الزرد الغدد التناسلية الأنسجة

إعداد على نطاق متوسط ​​من

إنشاء الزرد اليرقات "نموذج الحيوان" للتحقيق مثقبيه الكروزية حركية في فيفو

ذوبان الجليد واستزراع كريوبريسيرفينج خطوط الخلايا المورفولوجية

فحص لسلامة حاجز الدم في الدماغ في دروسوفيلا melanogaster

تحديد حجم الكبد في لارفال زيبرافيش باستخدام المجهر برايتفيلد

تطوير نموذج عدوى حمار وحشي لـ Clostridioides difficile

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

تشريح وكيمياء الهيستولوجية المناعية وتركيب أدمغة ذبابة الفاكهة اليرقية والبالغة لتصور الفص البصري

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