The authors wish to thank Drs. Sung-wan An and Mike O&#8217;Donnell for practical advice on establishing this assay, Dr. Chih-Jen Cheng for helpful discussions on the use of ion-specific electrodes, and Dr. Chou-Long Huang for his mentorship and support. This work was supported by the National Institutes of Health (K08DK091316 to ARR) and the American Society of Nephrology Gottschalk Award to ARR.

<ol>
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The authors have nothing to disclose.

A utilização do ensaio de Ramsay, em conjunto com eletrodos íon-específico, permite a medição das taxas de secreção de fluidos e fluxos de iões em insetos isolado Malpighi (renais) túbulos. Vinte ou mais túbulos pode ser ensaiada de cada vez, permitindo que um maior rendimento em comparação com o ensaio in vitro de indivíduo microperfused túbulos. Além disso, os eléctrodos específicos de iões permitir a determinação das concentrações de iões dentro do fluido que é segregado in situ, o que limita os erros que possam ser introduzidos na transferência dos pequenos volumes de fluido para um segundo aparelho. As etapas críticas no âmbito do protocolo incluem garantir que túbulos são dissecados sem rendas ou lágrimas (que fará com que o fluido a ser segregado da lágrima, ao invés do corte final do ureter), e garantir que o ISE está a funcionar bem, determinando sua inclinação prévia para iniciar o experimento. Mesmo com dissecações cuidadosas, não é incomum para túbulos ocasionais de falhar a secretar (até ~ 10% dos túbulosdissecado), mas se uma elevada proporção de túbulos não secretam isso pode ser por razões biológicas (ie., taxas muito baixas de secreção sob essa condição particular). Algumas armadilhas que podem levar a um ISE não trabalhar bem incluem: ionóforo vazando (que pode ser devido à quebra ponta do eletrodo ou silanização inadequada), ou a introdução de uma bolha de ar na ponta. Estes problemas podem ser determinadas através da visualização do eléctrodo sob um microscópio composto e geralmente requerem um novo enchimento ISE com ionóforo antes de prosseguir. Além disso, um ISE pode funcionar bem durante vários dias, ou mesmo semanas, mas vai eventualmente falhar, obrigando mais uma vez a construção de um novo ISE. É também extremamente importante que o prato de ensaio é cuidadosamente lavado no final da experiência, porque os sais residuais irá alterar a composição iónica e osmótica da solução salina de banho em experiências subsequentes. Em nossa experiência, o Na + ISE descrito neste protocolo é less estável do que o K + ISE, muitas vezes exibem deterioração da sua inclinação ao longo do curso do experimento. Com efeito, Jayakannan et ai. Descreveu a melhoria da Na + ISE baseadas no mesmo ionóforo com melhor selectividade e estabilidade. Seu cocktail incluído 3,5% 4- terc -butylcalix [4] areno-tetraetílico de ácido tetraacético e 95,9% de éter 2-nitrofenil octilo, e 0,6% de potássio tetraquis (4-clorofenil) borato 26. Uma vez que o experimentador é confortável a realização deste ensaio, muitas permutações podem ser introduzidos para investigar a biologia subjacente de transporte iónico epitelial. Por exemplo, os túbulos de diferentes genótipos podem ser examinadas para determinar o papel de um transportador ou de iões transepitelial canal específico no transporte de fluidos e de iões 14, ou de vias de sinalização extracelulares ou intracelulares que regulam estes transportadores ou canais 14,27. A composição gota banho pode ser modificado para olhar para opapel dos seus componentes específicos, tais como iões, glucose ou aminoácidos 9, 27,28 ou osmolaridade. Ou, em si hemolinfa obtido a partir de animais criados nas diferentes condições podem ser utilizadas em lugar da solução salina de banho de 16. Medicamentos, hormonas (ou seus segundos mensageiros) ou péptidos podem ser adicionados para determinar os seus efeitos 2,9,14,23,29. No protocolo de corrente, fluidos e de iões de fluxos do segmento principal são examinados, uma vez que o segmento inicial não secretam fluido 2, e o segmento inferior está fora da gota de banho. No entanto, o protocolo pode ser modificado para analisar a actividade dos segmentos iniciais ou inferiores 30. Repórteres transgênicos, como o aequorin transgene sensível ao cálcio, 5, ou o pH e de detecção de cloreto de ChlopHensor transgene 12, pode permitir correlação das concentrações intracelulares de íon com transepithelial fluxo. Da mesma forma, Efetova et. manipulação ai. demonstraram específico de células de níveis de cAMP utilizando optogenetic controle de um adenililciclase fotoactivo, demonstrando os efeitos diferenciais de cAMP em células estreladas e principais 31. Enquanto o protocolo atual está focada nos túbulos de Malpighi de adultos Drosophila melanogaster, as técnicas utilizadas foram aplicados em outros insetos, incluindo Rhodnius prolixus 21, Hemideina maori 24, e várias espécies de mosquitos 17, bem como sobre larval Drosophila túbulos 16. Do mesmo modo, além de outros iões Na + e K +, tais como H +, Ca 2+ ou de tetraetilamónio pode ser ensaiada 30,32, bem como toxinas, tais como salicilato de 33. Finalmente, a técnica de eléctrodo específico de iões também podem ser usadas para medir as concentrações de iões de mamífero em túbulos microperfused, obviando a necessidade de iões radiomarcados 34,35. Em resumo, a utilização de Ramsay quantodizer com eletrodos íon-específico permite a medição de fluidos transepithelial e fluxos de iões de isolado Drosophila melanogaster túbulos renais. Juntamente com as poderosas ferramentas genéticas de Drosophila disponíveis, os mecanismos moleculares de transporte iónico epitelial pode ser elucidado.

<html> Transporte de íons epitelial renal subjacente iono- organismal e osmoregulation. A Drosophila melanogaster Malpighian (renal) túbulo oferece uma oportunidade única para estudar os mecanismos moleculares de transporte de íons epitelial. Isto é devido à combinação dos poderosas da genética de Drosophila, emparelhado com a acessibilidade dos seus túbulos renais para estudo fisiológico. O ensaio de Ramsay, em homenagem ao pesquisador que foi pioneiro na técnica 1, mede as taxas de secreção de fluidos de túbulos de Malpighi isolados, e foi criada em Drosophila em 1994 pela Dow e colegas 2. Isso pavimentou o caminho para novos estudos utilizando Drosophila ferramentas genéticas, tais como o sistema GAL4-UAS 3,4, para definir específico de células vias de sinalização que regulam a secreção de fluidos. Um exemplo inclui a sinalização de cálcio, em resposta a uma hormona peptídica, 5, 6,7, entre muitos outros. ve_content &quot;&gt; uma combinação de técnicas genéticas e estudo fisiológico clássica demonstrou que a geração de urina na mosca ocorre através da secreção de um líquido rico em cloreto de potássio a partir do segmento principal do túbulo. Isto ocorre através da secreção transepitelial paralelo de catiões, principalmente K + Na +, mas também, através da célula principal, e Cl -. através da secreção de células estreladas 8-12 A capacidade de medir separadamente transepiteliais K + e Na + fluxos permite uma caracterização mais detalhada dos mecanismos de transporte do que a medição da secreção de fluido sozinho. Por exemplo, nos túbulos de Drosophila não estimuladas, a Na + / K + -ATPase ouabaína não tem efeito sobre a secreção de fluido 2, mesmo quando a sua absorção em células principais é inibida pelo inibidor de transportador aniónico taurocolato orgânica 13. No entanto, e Linton O&#39;Donnell mostrou que a ouabaína despolarizao potencial da membrana basolateral, e aumenta o fluxo de Na + 9. Como mostrado nos resultados representativos, nós replicado estes achados, e mostrou que K + fluxo é concomitantemente diminuiu 14; o aumento do fluxo de Na + e diminuição do K + fluxo têm efeitos opostos sobre a secreção de fluido, resultando em nenhuma alteração na secreção de líquido. Assim, existem duas resoluções para o &quot;paradoxo ouabaína,&quot; ou seja, a observação inicial de que a ouabaína não tem efeito sobre a secreção de fluido no túbulo Drosophila:. Em primeiro lugar, nos túbulos estimulada, o efeito de ouabaína na secreção de fluido não é aparente devido à sua absorção pelo transportador de aniões orgânicos 13; e, segundo, nos túbulos não estimuladas, ouabaína tem efeitos sobre transepithelial Na + e K + fluxo opostas, resultando em nenhuma alteração líquida na secreção de fluidos (ver resultados representativos e ref. 9). Portanto, o principal papel do Na + / K + -ATPase nos túbulos não estimuladas é o de diminuir a concentração intracelular de Na + para gerar um gradiente de concentração de Na + para o favorável -coupled processos de transporte através da membrana basolateral. Com efeito, medindo em separado de Na + e K + fluxos, nós demonstramos que túbulos que falta o co-transportador de mosca de sódio-potássio-2-cloreto de (CCAN) diminuíram K transepitelial + fluxo, sem diminuição adicional após a adição de ouabaína, e nenhuma alteração em transepitelial Na + fluxo 14. Estes resultados suportada nossa conclusão de que o Na + que entra na célula através da CCAN é reciclado através do Na + / K + -ATPase. Em outro exemplo, Ianowski et al., Observaram que a redução da concentração do banho de K + de 10 mM a 6 mM de K + diminuiu transepitelial fluxo transepitelial e aumentou o fluxo de Na + nos túbulos de Rhodnius prolixus, sem alteração líquida no fluido de secreção <sup&gt; 15. Efeitos diferenciais de fluxo de Na + e K + fluxo através dos túbulos de larvas também têm sido observadas em túbulos de Drosophila em resposta a diferentes dietas de sal 16 e em duas espécies de mosquitos em resposta à criação de salinidade 17. O maior desafio na medição de fluxo de iões transepitelial na preparação de ensaio Ramsay é a determinação das concentrações de iões dentro do fluido segregado. Este desafio foi cumprido com soluções diferentes, incluindo photometery chama 18, o uso de íons radioativos 19, e eletrônica sonda comprimento de onda espectroscopia por dispersão de 20. Estas técnicas exigem transferência da gota líquido secretado a um instrumento para a medição de concentrações de íons. Uma vez que o volume de fluido que é segregado pelo túbulo Drosophila não estimulado é pequena, tipicamente ~ 0,5 nl / min, isto representa um desafio técnico e também introduz erro se algum do fluido é segregadaperdeu por ocasião da transferência. Em contraste, a utilização de eléctrodos específicos de iões permite a medição da actividade de iões (a partir do qual a concentração de iões pode ser calculado) in situ. O protocolo corrente foi adaptada da utilizada por Maddrell e colegas para medir transepitelial K + fluxo através do túbulo Rhodnius usando valinomicina como o K + ionóforo 21, e também descreve a utilização de um -butylcalix 4- terc [4] areno-ácido tetraacético tetraetil éster à base de Na + eléctrodo específico de iões especï¿½ico caracterizado por Messerli et. al. 22. Eletrodos íon-específicas também têm sido usados ​​para medir concentrações de íons no líquido secretado por túbulos de Malpighi no ensaio Ramsay em 9,23 adulto e 16 larval Drosophila melanogaster, a Nova Zelândia Alpine Weta (Hemideina maori) 24 e 17 em mosquitos. Aqui, descrevemos em detalhes o uso do Ramsay comodizer para medir as taxas de secreção de fluidos em túbulos de Malpighi de Drosophila melanogaster, bem como o uso de eletrodos de íon-específico para determinar as concentrações de K + e Na + dentro do fluido secretado e, portanto, o cálculo dos fluxos de iões transepithelial. Uma visão geral do ensaio é fornecida na Figura 1. <img alt="figura 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> Figura 1. Esquema do Malpighian Palhinha eo Ramsay Ensaio com uso de íon-específicas eletrodos para medir concentrações de iões. Esta figura ilustra a configuração para o ensaio Ramsay. (A) Cada mosca tem quatro túbulos, um par de túbulos anterior e um par de túbulos posteriores, que flutuam na cavidade abdominal cercado por hemolinfa. Em cada par, as duas túbulos juntar-se no ureter, que, em seguida, esvazia-se a urina na junção do intestino médio e hindgut. Os túbulos são cegos-ended. A urina é gerado pelo segmento principal secretor de fluido (representado em vermelho), e flui para fora do ureter e no intestino. Após dissecção, a par dos túbulos é dissociada do intestino, ao seccionar o ureter. (B) A par dos túbulos é então transferida para uma gota de solução salina de banho dentro de uma cavidade do prato de ensaio. Um dos dois túbulos, referido aqui como o &quot;túbulo âncora&quot;, é enrolado em torno de um pino de metal e é inerte. A outra é o túbulo túbulo secretora. O segmento inicial (que não secretam fluido) e segmento principal do túbulo segregam permanecer dentro da gotícula de solução salina de banho. Íons e movimento de água da salina de banho e para o lúmen do túbulo do segmento principal, em seguida, avançar em direção ao ureter, como ocorreria in vivo. O segmento inferior (azul) está fora da salina de banho e, portanto, inertes. Uma vez que o ureter é cortado, o fluido segregado surge como uma gota a partir da extremidade de corte do ureter. Tele secretada gota de fluido aumenta ao longo do tempo como a secreção continua, e o seu diâmetro é medido usando um micrómetro ocular. Uma camada de óleo mineral evita a evaporação do fluido que é segregado. Os eletrodos de referência específicas e de iões de medir a concentração de íons do fluido secretado. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<table>
	<tbody>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Ellsworth Adhesives</td>
			<td>http://www.ellsworth.com/dow-corning-sylgard-184-silicone-encapsulant-0-5kg-kit-clear/</td>
			<td>May be purchased from multiple distributors</td>
		</tr>
		<tr>
			<td>Petri dish, polystyrene, 100 mm x 15 mm</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Petri dish, polystyrene, 35 mm x 10 mm</td>
			<td>Corning Life Sciences</td>
			<td>Fisher 08-757-100A</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Scalpel Handle #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Scalpel Blades #1</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>Specific brand is not important; use appropriate sharps precautions</td>
		</tr>
		<tr>
			<td>Needle, 30G x 1/2</td>
			<td>Becton Dickinson</td>
			<td>305106</td>
			<td>Use appropriate sharps precautions</td>
		</tr>
		<tr>
			<td>Minutien pins, black anodized, 0.15 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope with ocular micrometer</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>Specific brand is not important; this is given as an example</td>
		</tr>
		<tr>
			<td>Sheet of black stained glass, 3 mm (1/8 inch) thick</td>
			<td>Hobby shop</td>
			<td></td>
			<td>Example includes Spectrum Black Opal by Spectrum Glass (http://www.delphiglass.com/spectrum-glass/opalescent/spectrum-black-opal)</td>
		</tr>
		<tr>
			<td>Glass cutting tools (glass cutter, glass cutting pliers)</td>
			<td>Hobby shop</td>
			<td></td>
			<td>Examples include the Studio Pro Lightweight Running Pliers by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/pliers-nippers/studio-pro-lightweight-running-pliers) and the Studio Pro Brass Glass Cutter by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/glass-cutters/studio-pro-brass-glass-cutter). Use appropriate safety precautions when cutting glass</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary tube, unfilamented, GC120-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td>
			<td>Warner Instruments</td>
			<td>30-0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary tube, filamented, GC120F-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td>
			<td>Warner Instruments</td>
			<td>30-0044</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric acid, 70%</td>
			<td>Sigma</td>
			<td>438073</td>
			<td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines. Specific brand is not important</td>
		</tr>
		<tr>
			<td>Cimarec 7 in x 7 in hotplate</td>
			<td>Fisher</td>
			<td>11675911Q</td>
			<td>Specific brand is not important; caution when heated</td>
		</tr>
		<tr>
			<td>Selectophore dichlorodimethylsilane</td>
			<td>Sigma</td>
			<td>40136-1ML</td>
			<td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td>
		</tr>
		<tr>
			<td>Two-step vertical pipet puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>Other pipet pullers can be used; this is given as an example</td>
		</tr>
		<tr>
			<td>Glass petri dish, 150 mm diameter x 15 mm height</td>
			<td>Fisher</td>
			<td>08-748E</td>
			<td>Specific brand is not important; only one dish needed</td>
		</tr>
		<tr>
			<td>World Precision Instruments E210 1 mm micropipette storage jar</td>
			<td>Fisher</td>
			<td>50-821-852</td>
			<td>May be available from other distributors. Useful to have two jars. Note that although this jar is specified for 1 mm pipets, and the pipets used here are 1.2 mm, in our experience the 1 mm jar works best for the 1.2 mm pipets.</td>
		</tr>
		<tr>
			<td>Silica Gel, Tel-Tale Desiccant, indicating, 10-18 mesh</td>
			<td>Fisher</td>
			<td>S161-500</td>
			<td>Indicating silica useful for determining whether silica gel retains desiccating ability</td>
		</tr>
		<tr>
			<td>World Precision Instruments MicroFil, 34G</td>
			<td>Fisher</td>
			<td>50-821-914</td>
			<td>May be available from other distributors.</td>
		</tr>
		<tr>
			<td>1 ml syringe with luer lock</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td>May be available from other distributors.</td>
		</tr>
		<tr>
			<td>3 ml syringe with luer lock</td>
			<td>Becton Dickinson</td>
			<td>309657</td>
			<td>May be available from other distributors.</td>
		</tr>
		<tr>
			<td>D300 3-way stopcock with female luer lock inlet port, male luer outlet port with rotating collar and guard</td>
			<td>Cole-Parmer</td>
			<td>UX-30600-02</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Female Luer Locking Connector</td>
			<td>4 Medical Solutions</td>
			<td>ADC 9873-10</td>
			<td>Specific brand is not important; barbed end is ~4 mm at narrowest point and ~7 mm at widest point.</td>
		</tr>
		<tr>
			<td>Silicone Tubing I.D. x O.D. x Wall: 1/16 x 1/8 x 1/32 in. (1.59 x 3.18 x 0.79 mm)</td>
			<td>Fisher</td>
			<td>14-179-110</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>E-3603 tubing, I.D. x O.D.: 1/32 x 3/32 in</td>
			<td>Fisher</td>
			<td>14171208</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Modeling clay</td>
			<td></td>
			<td></td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Selectophore potassium ionophore I, cocktail B</td>
			<td>Sigma</td>
			<td>99373</td>
			<td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td>
		</tr>
		<tr>
			<td>Selectophore sodium ionophore X</td>
			<td>Sigma</td>
			<td>71747</td>
			<td>Sodium ionphore X = 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester</td>
		</tr>
		<tr>
			<td>Selectophore 2-nitrophenyl octyl ether</td>
			<td>Sigma</td>
			<td>73732</td>
			<td></td>
		</tr>
		<tr>
			<td>Selectophore sodium tetraphenylborate</td>
			<td>Sigma</td>
			<td>72018</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#39;s Drosophila medium</td>
			<td>Life Technologies</td>
			<td>21720024</td>
			<td></td>
		</tr>
		<tr>
			<td>High impedance electrometer</td>
			<td>World Precision Instruments</td>
			<td>FD223a</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode holder 1 mm with 45&#176; body, vented, with handle</td>
			<td>Warner Instruments</td>
			<td>64-1051</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode holder 1 mm with straight body, vented</td>
			<td>Warner Instruments</td>
			<td>64-1007</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>Warner Instruments</td>
			<td>64-1318</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulators, pair</td>
			<td>Leitz</td>
			<td></td>
			<td>Various brands/models will work; this is an example</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Technical Manufacturing Corporation</td>
			<td>81-334-03</td>
			<td>This is an example; any Faraday cage will work</td>
		</tr>
		<tr>
			<td>Single gooseneck fiberoptic light</td>
			<td>Nikon</td>
			<td></td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>mineral oil</td>
			<td>Fisher</td>
			<td>BP-2629</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>forceps, Dumont #5 with Biologie tip</td>
			<td>Fine Science Tool</td>
			<td>11295-10</td>
			<td>May be available from other distributors.</td>
		</tr>
	</tbody>
</table>

use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule

Modulação de transporte iónico epitelial renal permite que os organismos para manter a homeostase iónica e osmótica na face da variação das condições externas. A Drosophila melanogaster Malpighian (renal) túbulo oferece uma oportunidade única para estudar os mecanismos moleculares de transporte de íons epitelial, devido à genética poderosas deste organismo e da acessibilidade dos seus túbulos renais para estudo fisiológico. Aqui, nós descrevemos o uso do ensaio de Ramsay para medir as taxas de secreção de fluidos a partir de isolado de moscas túbulos renais, com a utilização de eléctrodos específicos de iões para medir as concentrações de sódio e potássio em que o fluido segregado. Este ensaio permite o estudo de fluidos e de iões transepitelial fluxos de ~ 20 túbulos de cada vez, sem a necessidade de transferir o fluido segregado a um aparelho separado para medir as concentrações de iões. Túbulos geneticamente distintas podem ser analisados ​​para avaliar o papel de genes específicos em processos de transporte. Além disso, o bathing salina pode ser modificado para analisar os efeitos das suas características químicas, ou drogas ou hormonas acrescentados. Em resumo, esta técnica permite a caracterização molecular dos mecanismos básicos de transporte epitelial de iões no túbulo Drosophila, bem como a regulação destes mecanismos de transporte.

As Figuras 7 e 8 demonstram que a utilização do ensaio de Ramsay com eléctrodos específicos de iões para medir as concentrações de K + e Na + pode distinguir geneticamente e farmacologicamente distintas K + e fluxos de Na +, a informação que não é capturada medindo as taxas de secreção de fluidos sozinho. Figura 7 mostra que a diminuição da secreção de fluido nos túbulos de moscas que transportam uma mutação nula homozigótica no CCAN é accionado por um decréscimo em K + de fluxo na mutantes (Figura 7A), enquanto que o Na + fluxo não é alterado (Figura 7B). A Figura 8 mostra que a Na + / K + -ATPase, a ouabaína, tem efeitos distintos em cada um dos três parâmetros medidos: em túbulos de tipo selvagem, taxas de secreção de fluido é inalterado (Figura 8A e 8B), o fluxo de Na + é aumentada (Figura 8A ), e fluxo de K + é devincado (Figura 8B). Além disso, a ouabaína não tem efeito no fluxo transepitelial K + nos túbulos NKCC mutantes nulos (Figura 8B). Em conjunto, estes dados indicam que a Na + / K + -ATPase fornece a força motriz para a absorção do K + através da CCAN, enquanto o Na + é reciclado através do Na + / K + -ATPase 14. Entre 13 e 18 túbulos foram analisados ​​em cada genótipo / condição; diferenças observadas foram estatisticamente significantes com p &lt;0,01 (ou menos) por não emparelhado, bicaudal teste t de Student. Desde ~ 15-20 túbulos podem ser facilmente analisados ​​no experimento de um único dia, os resultados mostrados aqui representam ~ 10 dias de experimentação. <img alt="Figura 7" src="/files/ftp_upload/53144/53144fig7.jpg" /> Figura 7. Fluid Secreção Taxa e Transepitelial K <strong&gt; + Flux estão diminuídos em Palhinha Carregar uma Homozygous Null Mutação em NKCC, While Na + Flux é Inalterado. (A) Taxa de fluido secreção (nl / min / túbulo) e K + fluxo (pmol / min / túbulo) foram medidos em túbulos de moscas de controle (wBerlin) ou a partir de moscas portadores de uma mutação nula homozigose no NKCC mosca (w; Ncc69 r2 / Ncc69 r2). n = 18 túbulos / genótipo. (B) taxa de secreção de fluidos (nl / min / túbulo) e Na + fluxo (pmol / min / túbulo) foram medidos a partir de moscas de controlo (wBerlin, n = 13 túbulos) ou a partir de moscas que transportam uma mutação nula homozigótica no NKCC mosca ( W; Ncc69 R2 / Ncc69 R2, n = 14 túbulos). Os valores apresentados são a média ± erro padrão da média (SEM). NS, não significativo; **, P &lt;0,01;***, P &lt;0,001; ****, P &lt;0,0001. Esta figura foi adaptado de Rodan et. al. 14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53144/53144fig7large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 8" src="/files/ftp_upload/53144/53144fig8.jpg" /> Figura 8. Na + / K + -ATPase Recicla Na + Entrando pela NKCC. (A) Taxa de secreção de fluidos (nl / min / túbulo) e fluxo de Na + (pmol / min / túbulo) foram medidos nos túbulos de moscas de controle (wBerlin) na ausência (n = 13 túbulos) ou na presença (n = 15 túbulos) de 100 uM de ouabaina, um inibidor de Na + / K + -ATPase. (B) taxa de secreção de fluidos (nl / min / túbulo) e K + </sup&gt; Fluxo (pmol / min / túbulo) foram medidos nos túbulos dissecados a partir de moscas de controlo (wBerlin), na ausência (n = 17 túbulos) ou na presença (n = 17 túbulos) de 100 uM de ouabaina. Fluxo de K + também foi medido em NKCC túbulos nulos na ausência ou na presença de ouabaina (w; Ncc69 R2 / Ncc69 R2, n = 16 - 17 túbulos / condição). Os valores apresentados são a média ± SEM. NS, não significativo; **, P &lt;0,01; ***, P &lt;0,001. Esta figura foi adaptado de Rodan et. al. 14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53144/53144fig8large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> <table><tbody><tr><td colspan="2"> Drosophila salina </td><td></td><td></td></tr><tr><td> Para 1L: </td><td></td><td></td><td></td></tr><tr><td colspan="4"> Pesar 3,6 g de glicose e coloque em um copo; co definitivancentration = 20 mM </td></tr><tr><td> Então adicione: </td><td></td><td></td><td></td></tr><tr><td> Componente </td><td> Volume (ml) </td><td colspan="2"> A concentração final (mM) </td></tr><tr><td> H2O </td><td> ~ 800 </td><td></td><td></td></tr><tr><td> 2,5 M de NaCl </td><td> 47 </td><td> 117,5 </td><td></td></tr><tr><td> KCl 1 M </td><td> 20 </td><td> 20 </td><td></td></tr><tr><td> 1 M de CaCl2 </td><td> 2 </td><td> 2 </td><td></td></tr><tr><td> 1 M de MgCl2 </td><td> 8,5 </td><td> 8,5 </td><td></td></tr><tr><td> 1 M de NaHCO3 </td><td> 10.2 </td><td> 10.2 </td><td></td></tr><tr><td> HEPES 1 M </td><td> 15 </td><td> 15 </td><td></td></tr><tr><td> 1 M NaH 2 PO 4 </td><td> 4.3 </td><td> 4.3 </td><td></td></tr><tr><td colspan="2"> Agita-se, ajustar o pH a 7,0 </td><td></td><td></td></tr><tr><td colspan="2"> Adicionar H 2 O para um volume final de 1L </td><td></td><td></td></tr><tr><td colspan="4"> Filtro de esterilizar; evitar autoclavagem (glicose vai caramelizar) </td></tr><tr><td> Armazenar a 4 ° C </td><td></td><td></td><td></td></tr><tr><td colspan="3"> Descartar se houver sinais de crescimento de bactérias ou fungos </td><td></td></tr></tbody></table> Tabela 1. Fazendo Drosophila salina. <table><tbody><tr><td> Meio de Schneider </td><td></td></tr><tr><td> Componente </td><td> Concentração (mM) </td></tr><tr><td> glicina </td><td> 3.33 </td></tr><tr><td> L-arginina </td><td> 2.3 </td></tr><tr><td> Ácido L-aspártico </td><td> 3.01 </td></tr><tr><td> L-cisteína </td><td> 0,496 </td></tr><tr><td> L-cistina </td><td> 0,417 </td></tr><tr><td> EU-ácido glutâmico </td><td> 5.44 </td></tr><tr><td> L-glutamina </td><td> 12.33 </td></tr><tr><td> L-histidina </td><td> 2.58 </td></tr><tr><td> L-isoleucina </td><td> 1.15 </td></tr><tr><td> L-leucina </td><td> 1.15 </td></tr><tr><td> Cloridrato de L-lisina </td><td> 9.02 </td></tr><tr><td> L-metionina </td><td> 5.37 </td></tr><tr><td> L-fenilalanina </td><td> 0,909 </td></tr><tr><td> L-prolina </td><td> 14.78 </td></tr><tr><td> L-serina </td><td> 2.38 </td></tr><tr><td> L-treonina </td><td> 2.94 </td></tr><tr><td> L-triptofano </td><td> 0.49 </td></tr><tr><td> L-tirosina </td><td> 2.76 </td></tr><tr><td> L-valina </td><td> 2.56 </td></tr><tr><td> β-alanina </td><td> 5.62 </td></tr><tr><td> CaCl2 </td><td> 5.41 </td></tr><tr><td> MgSO4 </td><td> 15.06 </td></tr><tr><td> KCl </td><td> 21.33 &lt;/ td&gt; </tr><tr><td> KH 2 PO 4 </td><td> 3.31 </td></tr><tr><td> NaCl </td><td> 36.21 </td></tr><tr><td> NaHCO3 </td><td> 4.76 </td></tr><tr><td> Na 2 HPO 4 </td><td> 4.94 </td></tr><tr><td> ácido a-cetoglutárico </td><td> 1.37 </td></tr><tr><td> D-glicose </td><td> 11.11 </td></tr><tr><td> ácido fumárico </td><td> 0,862 </td></tr><tr><td> ácido málico </td><td> 0,746 </td></tr><tr><td> ácido succínico </td><td> 0,847 </td></tr><tr><td> trealose </td><td> 5.85 </td></tr><tr><td> Yeastolate </td><td> 2,000 mg / L </td></tr></tbody></table> Tabela 2. Composição de meio de Schneider. <table><tbody><tr><td colspan="2"> Pré </td><td colspan="2"> Postar </td><td> Intervalo </td><td> Slope / decil </td></tr><tr><td> mM</td><td> mV </td><td> mM </td><td> mV </td><td> 15 - 150 (pré) </td><td> 52,8 </td></tr><tr><td> 15 </td><td> -28,4 </td><td> 15 </td><td> -29,4 </td><td> 15-150 (post) </td><td> 54,4 </td></tr><tr><td> 75 </td><td> 9.1 </td><td> 75 </td><td> 9.4 </td><td> 75 - 150 (pré) </td><td> 51 </td></tr><tr><td> 150 </td><td> 24,4 </td><td> 150 </td><td> 25 </td><td> 75-150 (post) </td><td> 52 </td></tr><tr><td> 200 </td><td> 30,6 </td><td> 200 </td><td> 31,9 </td><td> 150 - 200 (pré) </td><td> 49,6 </td></tr><tr><td></td><td></td><td></td><td></td><td> 150-200 (post) </td><td> 55,2 </td></tr><tr><td></td><td></td><td></td><td></td><td> Significar </td><td> 52,5 </td></tr></tbody></table> Tabela 3. Calibração de K </strong&gt; ISE + A K + ISE é calibrado em soluções contendo 15, 75, 150 e 200 mM de KCl, antes e depois da medição do fluido que é segregado experimental.; um exemplo de medições reais é mostrada nesta tabela. Cálculo da alteração no declive / decil na concentração de K + é ilustrado. Para o intervalo de 15-150 mV, inclinação / decil = mV para a queda de calibração a 150 mM - mV para a queda de calibração 15 mM. Para o 75 - intervalo 150, a diferença é dividida por 0,3 [log (150/75)]. Para a 150 - intervalo de 200, a diferença é dividida por 0,125 [log (200/150)]. Os seis pistas (três pré-experimento, três pós-experimento) são calculados em conjunto para obter a média inclinação / decil. Esta tabela foi adaptada de Rodan et. 14 al.

Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Este protocolo descreve a utilização do ensaio de Ramsay para medir as taxas de secreção de fluidos de Malpighi (túbulos renais) isolado a partir de Drosophila melanogaster. Além disso, a utilização de eléctrodos específicos de iões para medir as concentrações de sódio e potássio no líquido segregado, que permite o cálculo do fluxo de iões transepitelial, é descrito.

A utilização do Ramsay ensaio para medir secreção de fluidos e Ion Flux Preços no<em&gt; Drosophila melanogaster</em&gt; Tubule Malpighian

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

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Nanyang Technological University

Research

JoVE Journal

Biology

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

11.7K visualizações.  University of Texas Southwestern Medical Center.  Este protocolo descreve a utilização do ensaio de Ramsay para medir as taxas de secreção de fluidos de Malpighi (túbulos renais) isolado a partir de Drosophila melanogaster. Além disso, a utilização de eléctrodos específicos de iões para medir as concentrações de sódio e potássio no líquido segregado, que permite o cálculo do fluxo de iões transepitelial, é descrito. 

Vídeo: Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Técnicas de microinjeção para estudar na Mitose Drosophila melanogaster Embrião Sincicial

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Biologia

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP & FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fotobranqueamento Ensaios (FRAP &amp; FLIP) a Medida Dynamics Protein Chromatin em Viver Células-Tronco Embrionárias

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Injeção de insulina e Extração de hemolinfa para medir a sensibilidade à insulina em adultos Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

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 ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

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

Imaging Through the Pupal Case of Drosophila melanogaster

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

Imaging Through the Pupal Case of Drosophila melanogaster

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

Imagem Através do Pupal Caso de<em&gt; Drosophila melanogaster</em

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

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Quantitative Reação de Imunofluorescência Indireta para medir a variação nos níveis de proteína no centrossomas

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

PH intracelular de quantificação de óptica em Drosophila melanogaster túbulo de Malpighi epitélios com um indicador de pH fluorescente geneticamente codificado

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Infecção bacteriana e oral Shedding em Drosophila melanogaster.

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualizando e rastreando mRNAs endógenas em câmaras de ovo de Drosophila melanogaster ao vivo

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...