Este trabalho foi financiado pelo National Institutes of Health concede F32 NS078958 (DTB) e R01 AG033620 (BG).

1. Montar vôo Tester <ol><li> Cilindro vôo seguro para Anel Suporte 1 usando grampos cadeia. (Deixe cerca de 3 cm por baixo do cilindro para pesar prato.) (Nota: O cilindro de vôo que usamos é de 90 cm de comprimento, com um diâmetro de 13,5 cm.) </li><li> Insira pesar a cápsula com uma fina camada de óleo mineral por baixo do cilindro de voo. </li><li> Funil Segura para Anel Fique 2, utilizando uma braçadeira de anel e grampo garra. Ajustar a altura do funil de modo a que a parte inferior do funil é alinhada com a parte superior do cilindro de voo. (Nota: o diâmetro da ponta do funil deve ser menor do que o diâmetro exterior dos tubos de ensaio colocados nos tubos de descarga de modo a que os frascos não vai cair.) </li><li> Inserção do tubo de queda para o topo do funil e fixá-lo usando uma braçadeira garra. (Nota: Nós usamos um tubo de queda que é de 25 cm de comprimento Dropping voar contendo frascos dessa altura permite ejeção consistente de todas as moscas com força uniforme O diâmetro interno do tubo de queda deve ser sligh..tly maior do que o diâmetro externo do frasco para permitir que o frasco de cair livremente.) </li><li> Folha de corte (s) de poliacrilamida para o tamanho adequado. (Nota: Para ajudar na colocação e remoção da folha, a largura deve ser ligeiramente menor do que a circunferência interna do cilindro de voo). </li><li> Aplique uma fina camada de Emaranhado-Trap para a folha. Deixe descansar por 1 hora antes de usar. (Nota: Deixe espaço suficiente na parte superior e inferior da folha (aproximadamente 3 cm) não revestido para agarrar a folha para a inserção / remoção.) </li><li> Insira a folha de poliacrilamida no cilindro de vôo. </li><li> Monte a pista câmera usando suportes de pinho. (Nota: garantir que o fundo da pista pode suportar a câmera sem bloquear a lente Consulte a Figura 1B.). </li><li> Adicionar rolhas e parafuso no lugar. (Nota: coloque as rolhas em locais que permitam a câmera para ver toda a folha de plástico no modo panorâmico.) </li></ol> 2. Execute Experiment <ol> &lt;li&gt; Colete frascos de moscas a serem testadas. Para melhores resultados, usar não mais do que 20 moscas / frasco. </li><li> Bata suavemente voa para baixo do frasco, retire e insira em tubo de queda e liberar frasco. (Nota: O frasco cai no tubo de queda até atingir a abertura funil estreito Quando o frasco atinge o funil, as moscas são ejetados para dentro do cilindro de vôo.). </li><li> Levante o tubo de queda para remover o frasco vazio. (Nota: Vários frascos de moscas do mesmo grupo de teste pode ser analisada em uma única folha de poliacrilamida Nós achamos que até 200 moscas (10 frascos de 20 moscas cada) pode ser testada e fotografada prontamente em uma única folha.. </li><li> Retire a folha de plástico e coloque-o sobre uma superfície branca plana. (Nota: cartolina branca pode ser usado se bancadas são de cor escura.) </li><li> Monte a pista câmera sobre a folha de plástico. A câmara deverá ser suficientemente elevado acima da folha ter tanto a parte superior e inferior da folha, no campo de visão. </li><li> Deslize a câmera junto ªe pista, mantendo o botão &quot;capturar&quot; a adquirir uma imagem panorâmica. </li><li> O número de moscas que aterram no óleo podem ser contados manualmente para cada julgamento. </li><li> Repita os passos de 2,2-2,7 para todas as condições de um determinado experimento. As moscas podem ser removidas da folha entre cada ensaio. Alternativamente, pode ser usado várias folhas, com uma nova folha para cada julgamento. </li></ol> 3. Coleta de Dados <ol><li> Abrir arquivos de imagem usando o software ImageJ. </li><li> Corte imagens se necessário incluir apenas a área da superfície de aterragem. (Esta é a área revestida em emaranhado-Trap.) </li><li> Converter imagens de 8-bit em tons de cinza. </li><li> Criar um &quot;Threshold&quot; para filtrar o fundo branco. (Imagem → Ajustar → Threshold). </li><li> Defina os parâmetros para identificar cada mosca utilizando o menu &quot;analisar partículas&quot;. (Analisar → Analisar Partículas) Definir os parâmetros utilizados para identificar uma partícula. Com a nossa criada, nós achamos que utilizar umárea de 5-90 pixels 2 e uma circularidade de 0,4-1,0 irá identificar com precisão todas as amostras. </li><li> Medir a localização de cada mosca utilizando a lista gerada de coordenadas de cada partícula. A coordenada x em pixels pode ser convertida para centímetros para calcular a altura da aterragem. </li><li> Importe a tabela em uma planilha eletrônica (como o Microsoft Excel). </li></ol>

<ol>
	<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+behavior.">Genetic dissection of behavior.</a> Sci. Am. 229, 24-37 (1973).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J. Vis. Exp. , e2504 (2011).</li><li>de Vries, S. E., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Stimulation+of+Escape+Behavior+in+Drosophila+melanogaster.">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster.</a> J. Vis. Exp. , e50192 (2013).</li><li>Mundiyanapurath, S., Certel, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+aggression+in+Drosophila+(fruit+flies).">Studying aggression in Drosophila (fruit flies).</a> J. Vis. Exp. , e155 (2007).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J. Vis. Exp. , e3795 (2012).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> J. Vis. Exp. , e193 (2007).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann. N.Y. Acad. Sci. 1184, e1-e20 (2010).</li><li>Atkinson, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+separation+of+two+behavioral+phenotypes+by+a+mutation+affecting+the+promoters+of+a+Ca-activated+K+channel.">Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.</a> J. Neurosci. 20, 2988-2993 (2000).</li><li>Atkinson, N. S., Robertson, G. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+component+of+calcium-activated+potassium+channels+encoded+by+the+Drosophila+slo+locus.">A component of calcium-activated potassium channels encoded by the Drosophila slo locus.</a> Science. 253, 551-555 (1991).</li><li>Elkins, T., Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutation+that+eliminates+a+calcium-dependent+potassium+current.">A Drosophila mutation that eliminates a calcium-dependent potassium current.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 8415-8419 (1986).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>Fry, S. N., Rohrseitz, N., Straw, A. D., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+control+of+flight+speed+in+Drosophila+melanogaster.">Visual control of flight speed in Drosophila melanogaster.</a> J. Exp. Biol. 212, 1120-1130 (2009).</li><li>Mronz, M., Lehmann, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+free-flight+response+of+Drosophila+to+motion+of+the+visual+environment.">The free-flight response of Drosophila to motion of the visual environment.</a> J. Exp. Biol. 211, 2026-2045 (2008).</li></ol>

Os autores não têm conflitos de interesse de divulgar.

Usando os métodos descritos aqui, temos sido capazes de avaliar rapidamente o desempenho de vôo de um grande número de mutantes de Drosophila, proporcionando maior eficiência do que era possível anteriormente. Para nossos experimentos, nós rotineiramente separar machos e fêmeas e criá-los em baixa densidade (menos de 20 moscas / frasco) para limitar a agressão que possa danificar asas. Outra consideração importante é controlar adequadamente as diferenças de desempenho de vôo, devido a diferenças de fundo genético. Nós também achamos que ajuda a permitir que as moscas de um mínimo de 24 horas para se recuperar da anestesia com dióxido de carbono antes do teste de vôo. Alternativamente, as moscas podem ser anestesiados por exposição a temperatura fria (4 ° C) para permitir a recuperação mais rápida, sem potencialmente influenciar o comportamento. O passo limitante da taxa no presente protocolo é removendo as moscas da folha de plástico entre os ensaios. Um método para aumentar a eficiência é a utilização de um grande número of folhas simultaneamente, deixando de lado as folhas à medida que são utilizados e limpeza-los todos de uma vez após a coleta de dados. As moscas que caem para o fundo necessita de ser contado manualmente, uma vez que não irá ser incluída na folha. Ainda assim, isso é fácil em comparação com o cálculo manualmente altura da aterragem. A necessidade de reaplicar Emaranhado-Trap para cada folha pode variar dependendo da espessura do revestimento é. Em nossa experiência, uma folha indivíduo vai durar um mês antes é necessário um novo revestimento. Uma vantagem adicional de utilizar Emaranhado-Trap sobre o óleo mineral é a capacidade de recuperar as moscas ao vivo a partir da folha. Uma vez que as moscas simplesmente aderir à superfície do emaranhado-Trap em vez de tornar-se imerso, moscas individuais podem ser facilmente removidas. moscas &quot;Flightless&quot; que caem para a parte inferior também podem ser recuperados através da substituição do tabuleiro de óleo mineral com um frasco vazio. Nós acreditamos que a medição automatizada de comportamento de vôo descrito aqui fornece um númemero de vantagens em relação aos métodos anteriores, o que permite um maior nível de rendimento, reprodutibilidade e precisão para telas genéticos. Scoring automatizado também tem sido utilizada para aumentar o rendimento para os ensaios de comportamento, tais como o ensaio de anel 11. Além disso, a medição direta da altura pouso proporciona uma maior sensibilidade do que uma simples medição de aprovação / reprovação (% panfletos, etc.), O que nos permite detectar diferenças mais sutis no desempenho de vôo. O ensaio descrito aqui pode ser complementada por outras análises posteriores que medem aspectos mais complexos do comportamento de vôo, incluindo o controle visual da velocidade de vôo de 12 e as respostas de vôo livre ao movimento 13. Embora estes testes são mais demorada e não passíveis de telas genéticos de larga escala, eles podem ajudar a fornecer mais informações sobre a função de um determinado gene em uma resposta locomotor.

Drosophila tem sido utilizado para estudar a base genética de comportamento 1, e os investigadores desenvolveram um número de maneiras para analisar vários tipos de comportamento 2-6. As moscas têm sido particularmente úteis no fornecimento de modelos úteis de distúrbios neuromusculares 7. Um ensaio comum usado para estudar o comportamento locomotor é o desempenho de vôo. O testador vôo original é útil para identificar vôo mutantes defectivos e para a avaliação quantitativa da capacidade voo 1, mas tem várias deficiências que limitam a sua aplicação para as telas de alto rendimento: o uso de cilindros revestidos de petróleo é confuso e complicado, algumas funções, como o comprimento do cilindro e introdução de moscas para dentro do tubo com força variável reduzir a precisão quantitativa e é difícil de recuperar moscas vivo do testador. Para superar essas limitações, nós modificamos o testador vôo para incluir uma série de melhorias. Nós adicionamos uma &quot;gota tuser &quot;a introdução de moscas para eliminar a variabilidade entre experiências e os utilizadores. Usamos folhas de acrílico removível revestido com um adesivo que permite uma mais fácil limpeza e recuperação de moscas individuais. Temos aumentado o comprimento do tubo de voo para melhorar a exactidão quantitativa e fiabilidade. Finalmente , usamos uma câmera digital e software de imagem para calcular as alturas de pouso de moscas. Acreditamos que essas melhorias serão úteis para qualquer laboratório interessado na realização de telas genéticos de larga escala por defeitos no desempenho de vôo.

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an improved method for accurate and rapid measurement of flight performance in drosophila

Drosophila provou ser um sistema modelo útil para a análise de comportamento, incluindo o vôo. O testador vôo caindo envolvido inicial voa em uma revestido a óleo cilindro graduado; altura de desembarque prevista uma medida de desempenho de vôo, avaliando como moscas longe vai cair antes de produzir o suficiente impulso para fazer contato com a parede do cilindro. Aqui nós descrevemos uma versão atualizada do testador de vôo com quatro grandes melhorias. Primeiro, nós adicionamos um &quot;tubo de queda&quot; para garantir que todas as moscas entrar no cilindro de vôo a uma velocidade semelhante entre ensaios, eliminando a variabilidade entre os usuários. Em segundo lugar, substituiu o revestimento de óleo com folhas de plástico removíveis revestidas em Emaranhado-Trap, um adesivo projetado para capturar insetos vivos. Em terceiro lugar, nós usamos um cilindro de mais tempo para permitir a discriminação mais precisa da capacidade de vôo. Em quarto lugar, usamos uma câmera digital e software de imagem para automatizar a marcação de desempenho de vôo. Estas melhorias permitem o rapid, avaliação quantitativa do comportamento de vôo, útil para grandes conjuntos de dados e telas genéticos de larga escala.

<html> Figura 1A mostra um esquema da montagem testador vôo atualizado. Figura 1B ilustra o projeto de trilha permitem a câmera para tirar uma imagem panorâmica sem bloquear o campo de visão. Os resultados representativos são mostrados na Figura 2, em que o desempenho de voo de moscas mutantes Slowpoke, que têm um voo conhecidos defeito 8-10, são comparados com o tipo selvagem Canton-S voa. Controle voa consistentemente pousar perto do topo do cilindro, com muito pouca difusão entre os indivíduos e uma altura média de desembarque de 73 ± 2,0 cm. Em contraste, as moscas malandrinho exibir um spread muito mais variado de pouso e terra significativamente mais baixa, com média de 44 ± 4,1 cm. Todas as moscas foram 3 dias de idade, criados à temperatura ambiente (23 ° C). &quot;width =&quot; s/ftp_upload/51223/51223fig1.jpg 600px &quot;/&gt; Figura 1. Diagramas de vôo Tester, Camera, e Track. (A) Ilustração do estabelecido para o testador de vôo atualizado. Anel Suporte 1 mantém o cilindro vôo 90 cm de altura; Anel Fique 2 mantém o funil eo &quot;tubo de queda&quot; 10 centímetros de comprimento. (B) Diagrama de câmera ea trilha usada para produzir uma imagem panorâmica. A câmera deve ser apoiada pela pista sem obstruir a vista da lente. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51223/51223fig1highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada</a> . <img alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/51223/51223fig2highres.jpg" src="/files/ftp_upload/51223/51223fig2.jpg" width="600px" /> Figura 2. Os resultados representativos de um experimento de vôo amostra. Comparação da capacidade de vôo de três dias de idade Cacontrole nton-S voa para Slowpoke mutantes (slo TS1). (A) Tela de captura para as moscas slowpoke e controle exibindo as alturas de pouso de moscas individuais. Cada círculo azul representa o local de uma mosca individual. Estas alturas de aterragem são usados ​​para calcular a altura média de aterragem (B), bem como a distribuição geral (C) para cada genótipo. Moscas machos e fêmeas foram agrupados em cada amostra. As barras de erro representam o erro padrão da média. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51223/51223fig2highres.jpg" target="_blank">Clique aqui para ver a imagem ampliada</a> .

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An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Aqui nós descrevemos um método para a medição rápida e precisa do desempenho de vôo em Drosophila, permitindo seleção de alto rendimento.

Um método melhorado para rápido e preciso de medição de desempenho em vôo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

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Biology

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

11.7K visualizações.  University of Wisconsin-Madison.  Aqui nós descrevemos um método para a medição rápida e precisa do desempenho de vôo em Drosophila, permitindo seleção de alto rendimento. 

Vídeo: An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Um método simples para genotipagem Accurate

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Biologia

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

Um sistema de acoplamento magnético para Investigar controle de vôo visual e olfativo Mediated em Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

Visualmente Rastreamento Odor Mediated Durante o Voo em Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

Protocolo de treinamento de resistência e Ensaios de desempenho longitudinal para Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

Measurement of Lifespan in Drosophila melanogaster

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

Measurement of Lifespan in Drosophila melanogaster

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

Medição do Tempo de vida em<em&gt; Drosophila melanogaster</em

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

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

Um método inovador para exossomo Quantificação e Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

A medição da taxa metabólica em<em&gt; Drosophila</em&gt; Usando respirometria

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Um Método Seccionamento melhorado para amostras de tecidos de plantas recalcitrante

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

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

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

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

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

Determinação de área óptica transversal muscular dos músculos de voo indirecto adulto de Drosophila Melanogaster

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