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. Reagentes e Soluções <ol><li> Preparação do alimento mosca e mídia <ol><li> Mosca calor ingredientes de açúcar 11%, 2% de levedura autolisada, 5,2% de farinha de milho, agar de 0.79% w / v em água em uma placa fixada em 90 ° C. Agita-se regularmente até uma mistura ligeiramente densa homogénea. Preparar um volume final de 550 ml de alimento mosca para um total de 100 tubos de ensaio com 5 ml em cada mosca alimentos. </li><li> Remover a partir da fonte de calor, agita-se ocasionalmente até que o alimento tiver arrefecido até 80 ° C e adicionar 0,2% tegosept-metil 4-hidroxibenzoato de metilo Dissolveu-se em etanol a 95%. </li><li> Dividir a comida em dois volumes iguais. Adicionar 1,1 ml de 50 mM de rapamicina dissolvidos em etanol para um volume e 1,1 ml de etanol para o outro volume. Agita-se a rapamicina e as soluções de etanol para a comida. Certifique-se de que a temperatura cai para 50 ° C antes da adição de drogas. </li></ol></li><li> Tampão de isolamento <ol><li> Preparar 500 ml de tampão de isolamento [Manitol 225 mM, sacarose 75 mM, 10 mM de ácido 3- (N &lt;/ em&gt;-morfolino) propanossulf único (MOPS) e 1 mM de ácido etilenodiaminotetracético (EDTA), 2,5 mg / ml de albumina de soro bovino (BSA)] em água. </li><li> Ajustar o pH para 7,2 utilizando hidróxido de sódio. O pH inicial irá ser ácido. </li><li> Use garrafas de armazenamento filtro estéril com poros de 0,22 um para esterilização da solução e armazená-lo a 4 ° C. </li></ol></li><li> Tampão de Lavagem <ol><li> Preparar 500 ml de tampão de lavagem [Manitol 225 mM, sacarose 75 mM, KCl 10 mM, Tris HCl 10 mM e 5 mM de KH 2 PO 4] em água. </li><li> Ajustar o pH para 7,2 utilizando hidróxido de sódio. O pH inicial irá ser ácido. </li><li> Use garrafas de armazenamento filtro estéril com poros de 0,22 um para esterilização da solução e armazená-lo a 4 ° C. </li></ol></li></ol> 2. Criação de as estirpes de Drosophila <ol><li> Para controlar a densidade larval durante o desenvolvimento, coloque 25 pares de pais em um frasco de cultura e permitir-lhes a pôr ovos durante 48 horas. </li> &lt;li&gt; Remova os pais depois de 48 horas para regular a densidade dos ovos. </li><li> Repita os passos 2.1 e 2.2 utilizando a prole F1 emergentes, de modo duas gerações deste controle de densidade é atingida. </li><li> Para a coleta de adultos, anestesiar as moscas que utilizam o dióxido de carbono (CO 2) e separados por sexo. <ol><li> Usar um único regulador de fase pura para entrega de CO 2 a partir de um tanque de alta pressão com um fluxo contínuo de 5 psi. Para evitar a electricidade estática, utilizar um tubo de plástico para catalisar a CO 2 em um frasco de 500 ml com água de filtragem. </li><li> Usar uma rolha de borracha com um orifício para selar o frasco. Utilize tubos de plástico para ligar a abertura lateral do balão a uma almofada de CO 2. </li><li> Coloque as moscas na almofada para não mais de 10-15 min. Deixa-se recuperar da anestesia durante 24 h antes de colocá-los em condições experimentais. </li></ol></li><li> Para gerar metabólitos suficientes para fracções enriquecidas mitocondriais, use 300 moscas por amostra. Aqui, use as fêmeas, mas gendeR pode ser diferente em outras experiências. Transferir 150 moscas caseiras 1 L gaiola demografia. Permitir o acesso a 5 ml de alimentos num frasco de mosca. </li><li> Transferir os restantes 150 moscas para outro 1 L gaiola. Não overpopulate gaiolas com mais de 150 moscas. </li><li> Use seis amostras idênticas, por condição experimental. Desde extratos de animais inteiros render mais metabolitos, para gerar uma amostra de toda animais isolados, transferir 50 fêmea voa para uma gaiola. Como para os isolados mitocondriais, utilize seis amostras idênticas, por condição experimental. </li><li> Coloque gaiolas a 25 ° C, com ciclo de 12 horas de luz e 12 horas escuro. </li><li> Fornecer fresco a cada 2 ou 3 dias num frasco com 5 ml de alimento para manter a qualidade dos alimentos. </li><li> Manter moscas em comida para 10 dias. Este passo é necessário para o tratamento de rapamicina 7. Outros tratamentos ou condições serão diferentes. </li></ol> 3. Isolamento de fracções mitocondriais <ol><li> Dump voa de uma gaiola (150 moscas) em umvidro-Teflon homogeneizador Dounce cheio com 1 ml de tampão de isolamento arrefecido. </li><li> Homogeneizar movendo o pilão cima e para baixo por 15 tacadas. Mantenha a argamassa em gelo para manter mitocôndrias intactas. </li><li> Transferência homogeneizado para um tubo de 1,5 ml e centrifugar a 300 xg durante 5 min a 4 ° C. </li><li> Tome-se o sobrenadante e centrifuga-se a 6000 xg durante 10 min a 4 ° C para enriquecer para mitocôndrias. </li><li> Como o sobrenadante contém a fracção citosólica, desprezar o sobrenadante ou usá-la para experiências alternativas. Ressuspender o sedimento em 300 ul de tampão de lavagem. </li><li> Repita os passos de 3,1-3,5 para a segunda gaiola. Combinam-se as peletes ressuspensas de duas gaiolas num tubo de microcentrífuga criogénico. </li><li> Centrifugar a 6.000 xg durante 10 min a 4 ° C. </li><li> Descartar o sobrenadante e congelar o Flash-o sedimento em azoto líquido. </li><li> Armazenar as fracções enriquecidas mitocondriais a -80 ° C ou temperatura inferior. <ol><li> Como alternativa, para animais pré inteiroparation, colocar os adultos em um tubo de microcentrífuga criogénico. Flash congelar o frasco em azoto líquido e armazenar adultos a -80 ° C ou temperatura inferior. </li></ol></li></ol>

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Os passos mais críticos neste protocolo são: 1) criação de moscas suficientes no espaço abundante. É muito importante para as gaiolas não overpopulate demografia com mais de 150 moscas cada; 2) mudando a comida das gaiolas com freqüência para evitar a concorrência alimentar e estresse nutricional; e 3) a manutenção de todas as amostras a 4 ° C para assegurar a integridade durante o isolamento da fracção mitocondrial. Também é recomendado para relaxar o tampão de isolamento, o tampão de lavagem, e o ho...

O objetivo deste procedimento é desenvolver frações mitocondriais enriquecidos que produzem metabólitos mitocondriais suficientes para metabolômica estudos utilizando Drosophila melanogaster. Em nossa experiência, metabolômica análise usando métodos de extração celular inteiras são incapazes de detectar mudanças sutis metabólitos mitocondriais em Drosophila. No entanto, fracionamento mitocondrial antes da análise metabolômicas aumenta a sensibilidade para identificar mudanças de metabolitos mitocondriais. As mitocôndrias são organelas celulares responsáveis ​​pelo fornecimento de 90% da energia que as células precisam para a função normal 1. Nos últimos anos, tem sido reconhecido que as mitocôndrias desempenhar um papel muito mais dinâmico na função celular e do organismo do que simplesmente produzir trifosfato de adenosina (ATP), e são agora reconhecidos como hubs para a regulação da homeostase metabólica 2,3. As mitocôndrias são o resultado de um processo em que endosymbiotic dislinhagens microbianas tinct fundiu ~ 1,5 bilhões de anos atrás 4. Como as mitocôndrias evoluíram para verdadeiras organelas, genes da endosymbiont foram incorporados no genoma nuclear emergente. Em animais, hoje, cerca de 1.500 proteínas mitocondriais são codificados nuclear enquanto 37 genes permanecem na mtDNA, 13 dos quais codificam proteínas mitocondriais que são subunidades dos complexos de enzimas de fosforilação oxidativa 5. A coordenação entre as mitocôndrias e compartimentos nucleares é necessária para manter a função mitocondrial adequada. Usando os métodos descritos aqui, fomos capazes de detectar alterações metabólicas mitocondriais em Drosophila que resultam da manipulação da coordenação entre os genomas mitocondriais e nucleares. Nós usamos uma linhagem de Drosophila em que mtDNA de suas espécies irmãs D. simulans foi colocada sobre um único D. melanogaster fundo nuclear 6. Este mitonuclear &#39;interrompido&#39;genótipo foi comparado com o genótipo mitonuclear &quot;nativo&quot;, ou co-evoluiu de D. melanogaster transportando a mesma com o seu genoma nuclear nativa D. melanogaster mtDNA. O D. melanogaster e D. simulans mtDNAs diferem por ~ 100 aminoácidos e&gt; 500 substituições sinônimas que afetam mitonuclear 7,8 comunicação. Geramos extratos mosca integrais e extratos enriquecidos mitocondriais para estudar mudanças de metabolitos em resposta ao estresse farmacológico. Aqui nós mostramos que, ao usar frações mitocondriais enriquecido detectamos mudanças acentuadas em metabólitos mitocondriais entre o genótipo nativa, co-evoluído levando a D. mtDNAs melanogaster e as genótipo rompidas transportando D. simulans mtDNA. Em contraste, as modificações do metabolito entre estes dois genótipos são subtis usando métodos normais que utilizam extracto mosca todo. Por conseguinte, este método forneceu um caminho para entender como mtDNAs mediar alterações mitocondriais emresposta a diferentes drogas.

<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.

Usando o protocolo acima referido, foi realizada a análise fracções enriquecidas em metabolômico mitocondriais e extractos de animais inteiros para testar o efeito do fármaco rapamicina em mtDNAs divergentes 7. Obtivemos 200 uM de rapamicina por dissolução da droga no alimento mosca. As moscas foram expostos a rapamicina durante 10 dias. Os metabolitos de extractos de mosca inteiras e extractos de mitocondriais foram obtidos utilizando um espectrómetro de massa de...

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.

Preparação de mitocondriais frações enriquecidas para Análise metabólica em<em&gt; Drosophila</em

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

preparation-mitochondrial-enriched-fractions-for-metabolic-analysis

Research

JoVE Journal

Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

9.5K visualizações.  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. 

Vídeo: 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.

Análise de Zebrafish larvas Músculo Esquelético Integrity com 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 ...

Biologia do Desenvolvimento

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).

Preparação em média<em&gt; Drosophila</em&gt; Extractos de embrião para experiências proteômicas

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.

Dissecção e imunofluorescência coloração de cogumelo corpo e fotoreceptor neurônios em cérebros adultos Drosophila melanogaster

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.

Preparando a desenvolver tecido olfativo periférico para Molecular e análise imuno-histoquímica em Drosophila

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.

Preparação de amostras de larvas de Drosophila por cromatografia gasosa / espectrometria de massa (GC-MS)-com base em metabolómica

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;

Dissecação da Retina Pupal drosófila para imuno-histoquímica, análise ocidental e isolamento de RNA

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.

Imagens ao vivo e análise de contrações musculares no embrião de Drosophila

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.

Descongelamento, cultivo e Cryopreserving linhas de células de Drosophila

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.

Preparação de Drosophila Larval e Pupal Testes para Análise de Divisão Celular em Tecido Vivo e Intacto

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.

Uso de Bisection para reduzir DNA mitocondrial no Oócito Bovino

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