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

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.

<html> 1. Forberedelse dissektion, kalibrering og Assay Retter Bemærk: I dette trin, er tre petriskåle af plast foret med silikone elastomer fremstillet: én for dissektion, en til udførelse af Ramsay assay (&quot;assay skål&quot;), og én til at udføre kalibrering. Disse retter er genbruges fra eksperiment til eksperiment, og dermed dette trin skal kun gentages, hvis en skål pauser. Et billede af assayet skålen er vist i figur 2. <p class="jove_content" fo:keep-toge...

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Brugen af ​​Ramsay assay sammen med ionspecifikke elektroder, tillader måling af væske sekretionshastigheder og ionstrømme i isoleret insekt Malpighian (nyre) tubuli. Tyve eller flere tubuli kan analyseres ad gangen, giver højere gennemløb i forhold til assayet individuelle in vitro microperfused tubuli. Desuden ionspecifikke elektroder tillader bestemmelse af ionkoncentrationer i den udskilte væske i situ, begrænse fejl, der kan indføres i overførsel af små volumener af væske t...

<html> Renale epitel iontransport ligger til grund organismal iono- og osmoregulering. Drosophila melanogaster Malpighian (renal) tubulus tilbyder en enestående mulighed for at studere de molekylære mekanismer i epitelial ion transport. Dette skyldes kombinationen af de stærke genetik Drosophila, parret med tilgængeligheden af sine nyretubuli til fysiologisk undersøgelse. Ramsay assay, opkaldt efter den undersøger, som banebrydende teknik 1, måler væskesekretion satser fra isolerede Malpighian tubuli, og blev etableret i Drosophila i 1994 af Dow og kolleger 2. Dette banede vejen for yderligere undersøgelser ved hjælp af Drosophila genetiske værktøjer, såsom GAL4-UAS systemet 3,4, for at definere celle-specifikke signalveje regulerer væske sekretion. Et eksempel omfatter calcium signalering som respons på et peptidhormon 5, blandt mange andre 6,7. ve_content &quot;&gt; en kombination af genetiske teknikker og klassiske fysiologiske undersøgelse har vist, at urin generation i fluen sker gennem sekretion af et kaliumchlorid-rige væske fra hovedsegmentet af tubuli. Dette sker via den parallelle transepitel sekretion af kationer, primært K + men også Na +, gennem den primære celle og Cl -. sekretion gennem stjerneformet cellen 8-12 Evnen til separat at måle transepiteliale K + og Na + flux giver en mere detaljeret karakterisering af transportmekanismer end måling af væske sekretion alene. For eksempel i ustimulerede Drosophila tubuli, Na + / K + -ATPase-inhibitoren ouabain har ingen effekt på fluid sekretion 2, selv når dets optagelse i celler vigtigste inhiberes af organisk aniontransporter inhibitor taurocholat 13. Imidlertid Linton og O&#39;Donnell viste, at ouabain depolarisererden basolaterale membran potentiale, og øger Na + flux 9. Som vist i Repræsentative resultater, vi gentaget disse resultater, og viste, at K + flux er samtidig faldet 14; den øgede Na + flux og faldt K + flux har modsatrettede virkninger på fluid sekretion, hvilket resulterer i nogen nettoændring i sekretion. Der er således to beslutninger til &quot;ouabain paradoks&quot;, dvs. den oprindelige iagttagelse, at ouabain har nogen virkning på fluid sekretion i Drosophila tubulus:. Dels i stimulerede tubuli, virkningen af ouabain på fluid sekretion fremgår ikke på grund af dens optagelse af organisk aniontransporter 13; og for det andet i ustimulerede tubuli, ouabain har modstående virkninger på transepitelialt Na + og K + flux, hvilket resulterer i nogen nettoændring i fluid sekretion (se Repræsentative resultater og ref. 9). Derfor er den primære rolle af Na + / K + ATPase i ustimulerede tubuli er at sænke intracellulære Na + koncentration til at frembringe en positiv koncentrationsgradient for Na + -coupled transportprocesser på tværs af basolaterale membran. Faktisk ved separat måling Na + og K + fluxe, vi påvist, at tubuli mangler fluen natrium-kalium-2-chlorid cotransportøren (NKCC) er faldet transepitelial K + flux, uden yderligere fald efter ouabain Desuden, og ingen ændring i transepitel Na + flux 14. Disse resultater støttede vores konklusion, at Na + ind i cellen gennem NKCC recirkuleres gennem Na + / K + -ATPase. I et andet eksempel Ianowski et al. Observerede, at en sænkning bad K + koncentration fra 10 mM til 6 mM reduceret transepitheliale K + flux og forøget transepitel Na + flux i tubuli fra Rhodnius prolixus, med nogen nettoændring i fluid sekretion <sup&gt; 15. Forskellige virkninger på Na + flux og K + flux på tværs af larver tubuli er også blevet observeret i Drosophila tubuli som reaktion på varierende salt kost 16 og i to myg arter som reaktion på opdræt saltholdighed 17. Den største udfordring i målingen af ​​transepitelial ion flux i Ramsay assay forberedelse er bestemmelsen af ​​ion koncentrationer inden det udskilte væske. Denne udfordring er blevet mødt med varierende løsninger, herunder flamme photometery 18, anvendelse af radioaktive ioner 19, og elektron sonde bølgelængde dispersivt spektroskopi 20. Disse teknikker kræver overførsel af den udskilte væske fald til et instrument til måling af ionkoncentrationer. Da mængden af væske, der udskilles af den ustimulerede Drosophila tubulus er lille, typisk ~ 0,5 nl / min, udgør det en teknisk udfordring, og også indfører fejl, hvis nogle af de udskilte væsketabt ved overførsel. I modsætning hertil anvendelsen af ionspecifikke elektroder tillader måling af ion aktivitet (hvorfra der kan beregnes ionkoncentration) in situ. Den nuværende protokol blev tilpasset fra den, der anvendes af Maddrell og kolleger til at måle transepitel K + flux på tværs af Rhodnius tubulus hjælp valinomycin som K + ionofor 21 og beskriver også anvendelsen af en 4-tert -butylcalix [4] aren-tetraeddikesyre tetraethylester-baserede Na + -specifik ion-specifik elektrode kendetegnet ved Messerli et. al. 22. Ion-specifikke elektroder er også blevet anvendt til at måle ion koncentrationer i væske, der udskilles af Malpighian tubuli i Ramsay assay voksne 9,23 og larver 16 Drosophila melanogaster, New Zealand Alpine Weta (Hemideina Maori) 24 og myg 17. Her beskriver vi i detaljer brugen af ​​Ramsay somsige at måle fluid sekretionshastigheder i Malpighian tubuli fra Drosophila melanogaster, samt anvendelsen af ionspecifikke elektroder til at bestemme koncentrationerne af K + og Na + i den udskilte væske og dermed beregningen af transepitelialt ionstrømme. En oversigt over assayet er tilvejebragt i figur 1. <img alt="Figur 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> Figur 1. Skematisk af Malpighian tubuli og Ramsay-analysen med brug af Ion-specifikke elektroder til måling ionkoncentrationer. Denne figur illustrerer opsætningen for Ramsay analysen. (A) Hver flue har fire tubuli, et par anteriore tubuli og et par posteriore tubuli, der flyder i bughulen omgivet af hæmolymfe. I hvert par, de to tubuli deltage i urinlederen, som derefter tømmer urinen ved krydset af mellemtarmen og hindgut. Tubuliene er blinde ender. Urin frembringes af fluid-udskillende hovedsegmentet (vist med rødt) og strømmer mod ureter og ud i tarmen. Efter dissektion bliver tubulus parret adskilt fra tarmen ved transecting ureter. (B) Parret af tubuli overføres derefter til en dråbe saltvand badning inden for en brønd i assay skålen. En af de to tubuli, der er nævnt her som &quot;anker tubulus,&quot; er viklet omkring en metalstang og er inert. Den anden tubulus er udskillende tubulus. Den indledende segment (som ikke udskiller væske) og vigtigste segment af secernerer tubulus forbliver inden for dråbe badning saltvand. Ioner og vand træk fra badning saltvand og ind i tubulus lumen de vigtigste segment, og derefter bevæge sig mod ureter, som ville forekomme in vivo. Den nederste segment (blå) er uden for badning saltvand og derfor inaktivt. Da ureter skæres, fremgår det secernerede fluid som en dråbe fra den afskårne ende af ureter. Than udskilte væske dråbe forstørrer, efterhånden som sekretion fortsætter, og dens diameter er målt ved anvendelse af en okulær mikrometer. Et lag af mineralolie forhindrer fordampning af det udskilte væske. Reference- og ion specifikke elektroder måler ion koncentrationen af det udskilte væske. <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">Klik her for at se en større version af dette tal.</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

Modulation af renal epitelial iontransport tillader organismer for at opretholde ioniske og osmotisk homeostase i ansigtet af varierende ydre forhold. Drosophila melanogaster Malpighian (renal) tubulus tilbyder en enestående mulighed for at studere de molekylære mekanismer i epitel iontransport, på grund af de kraftige genetik på denne skadegører, og tilgængeligheden af sine nyretubuli til fysiologisk undersøgelse. Her beskriver vi anvendelsen af ​​Ramsay assay til måling af væske sekretionshastigheder fra isoleret flyve nyretubuli, med anvendelse af ionspecifikke elektroder til måling af natrium og kalium koncentrationer i den udskilte væske. Dette assay muliggør undersøgelse af transepitel væske og ionstrømme af ~ 20 tubuli ad gangen, uden at det er nødvendigt at overføre den udskilte væske til en separat apparat til måling af ionkoncentrationer. Genetisk distinkte tubuli kan analyseres for at vurdere rollen af ​​specifikke gener i transportprocesser. Derudover bathing saltvand kan ændres til at undersøge virkningerne af sine kemiske egenskaber, eller narkotika eller hormoner tilføjet. Sammenfattende denne teknik tillader molekylær karakterisering af grundlæggende mekanismer i epitel iontransport i Drosophila tubulus, samt regulering af disse transportmekanismer.

Figur 7 og 8 viser, at brugen af Ramsay assay med ionspecifikke elektroder til måling af K + og Na + koncentration kan skelne genetisk og farmakologisk særskilte K + og Na + flusmidler, oplysninger, der ikke fanget ved måling fluid sekretionshastigheder alene. Fig 7 viser, at faldet fluid sekretion i tubuli mod fluer bærer en homozygot nul mutation i NKCC drives af et fald i K + flux i mutanterne (Figur 7A), men...

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

Denne protokol beskriver anvendelsen af Ramsay assay til måling af væske sekretionshastigheder fra isolerede Malpighian (renal) tubuli fra Drosophila melanogaster. Desuden anvendelsen af ​​ionspecifikke elektroder måler natrium og kalium koncentrationer i den udskilte væske, tillader beregning af transepitel ion flux, er beskrevet.

Anvendelse af Ramsay-analysen til at måle Fluid sekretion og Ion Flux priser i<em&gt; Drosophila melanogaster</em&gt; Malpighian tubulus

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

Research

JoVE Journal

Biology

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

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 ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

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

Photobleaching Assays FRAP & 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 ...

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

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 ...

Measurement of Lifespan in Drosophila melanogaster

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Måling af Levetid i<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

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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

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Imaging Gennem puppe Case of<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

Kvantitativ immunofluorescensassay at måle variationen i proteinniveauer på centrosomer

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

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

Optisk kvantificering af intracellulære pH i Drosophila melanogaster Malpighian tubulus epitheler med en fluorescerende genetisk kodet pH indikator

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

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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 ...