Name: a microfluidic platform for longitudinal imaging in caenorhabditis elegans
Uploaded: 2018-05-02T13:30:00
Description: Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

Denne forskningen ble støttet av National Science Foundation bevilgninger PHY-1205494 og kalt MCB-1413134 (EL) og National Research Foundation av Korea grant 2017R1D1A1B03035671 (KSL).

Protokollen nedenfor bruker WormSpa5, en tidligere beskrevet microfluidic enhet for langsgående avbilding av ormer. Fabrikasjon av WormSpa (starter med CAD-filer som kan oppnås fra forfatterne på forespørsel) er enkelt men krever noen kompetanse. I de fleste tilfeller kan lett fabrikasjon gjøres ved en kjerne anlegget eller kommersielt selskap som tilbyr slike tjenester. Når fabrikere enheten, må du angi at høyden på funksjonene er 50 µm.
1. eksperimentelle opps...

<ol>
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Wild-type growth and reproduction.</a> Dev Biol. 51 (1), 23-33 (1976).</li><li>Chalancon, G., 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=Interplay+between+gene+expression+noise+and+regulatory+network+architecture.">Interplay between gene expression noise and regulatory network architecture.</a> Trends Genet. 28 (5), 221-232 (2012).</li><li>Sanchez, A., Choubey, S., Kondev, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+noise+in+gene+expression.">Regulation of noise in gene expression.</a> Annu Rev Biophys. 42, 469-491 (2013).</li><li>Norman, T. M., Lord, N. D., Paulsson, J., Losick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+Switching+of+Cell+Fate+in+Microbes.">Stochastic Switching of Cell Fate in Microbes.</a> Annu Rev Microbiol. 69, 381-403 (2015).</li><li>Gardner, T. S., di Bernardo, D., Lorenz, D., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+genetic+networks+and+identifying+compound+mode+of+action+via+expression+profiling.">Inferring genetic networks and identifying compound mode of action via expression profiling.</a> Science. 301 (5629), 102-105 (2003).</li><li>Samuelson, A. V., Carr, C. 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Microfluidic verktøy gir flere fordeler i å studere ormer. Bildebehandling i en PDMS tilbyr enhet høyere tenkelig kvalitet sammenlignet med en standard NGM agar plate. Flere bilder kan hentes fra en enkelt orm, kontrast tradisjonelle metoder der dyr er plukket fra platen og montert på en microscope skyve for bildebehandling. I tillegg kan microenvironment som ormer bor holdes konstant eller modulert som ønsket, tillater presis kartlegging sammensetningen av miljøet og svaret av dyrene.
H...

Endringer i miljøforhold kan føre til aktivering av genetiske programmer ledsaget av induksjon og undertrykkelse av uttrykk for bestemte gener1,2. Endringene kinetic kan være variabel blant vev i de samme dyrene og mellom forskjellige dyr. Studier av genetiske programmer derfor kaller for metoder som tillater langsgående imaging enkelte dyr og gi nøyaktig dynamisk kontroll av miljøforhold.
De siste årene, har microfabricated fluidic enheter blitt brukt til å studere mange aspekter av respons og atferd i små dyr, inkludert ormer, fluer, vann bjørn og mer3,4,5,6, 7. Programmene omfatter, for eksempel dyp phenotyping, optogenetic innspillingen neuronal aktivitet som svar på kjemiske stimuli, og sporing av motorisk atferd som bevegelse og pumping8,9,10 , 11.
Microfluidic tilnærminger har mange egenskaper som kan ha nytte langsiktige longitudinelle imaging reaksjon på miljømessige signaler, inkludert nøyaktig dynamisk kontroll av den lokale microenvironment, fleksible design som gjør at vedlikehold av enkelte dyr i egne værelser og gunstige attributter for bildebehandling. Men opprettholde dyr i en microfluidic kammer lenge med minimal ugunstig innvirkning på deres godt vesener er en utfordring, som krever spesiell omsorg i utformingen av microfluidic enheten samt gjennomføring av eksperimentet.
Her viser vi bruk av WormSpa, en microfluidic enhet for langsgående avbildning av Caenorhabditis elegans. 5 individuelle ormer er begrenset i kamre. Konstant lav flyt av væske og bakteriell suspensjon garanterer at ormer er velfødde og tilstrekkelig aktiv for å opprettholde god helse og lindre stress, og strukturen i kamrene kan ormer å legge egg. Enkelheten av design og drift av WormSpa bør tillate forskere uten tidligere erfaring i microfluidics å innlemme denne enheten i sin egen forskningsplaner.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">WormSpa</td>
			<td style="width:123px;">N/A</td>
			<td style="width:344px;">N/A</td>
			<td style="width:329px;">The CAD file for WormSpa is available from the Levine lab.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Compound Microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver Z1</td>
			<td>An inverted fluorescence microscope with a motorized stage</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe Pump</td>
			<td>New Era Pump Systems</td>
			<td>NE-501</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tubing</td>
			<td>SCI Scientific Commodities Inc.</td>
			<td>BB31695-PE/5</td>
			<td>0.034&#8221; (0.86 mm) I.D. x 0.052&#8221; (1.32 mm) O.D</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe Tip</td>
			<td>CMLsupply</td>
			<td>901-20-050</td>
			<td>20 Gauge x 1/2&#8221; blunt tip stainless steel canula</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe Filter</td>
			<td>PALL</td>
			<td>4650</td>
			<td>Acrodisc 32 mm Syringe Filter with 5 um Supor Membrane</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Syringe</td>
			<td>Qosina</td>
			<td>C3307</td>
			<td>10 mL Male Luer Lock Syringe</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">3 Way Valve</td>
			<td>ColeParmer</td>
			<td>FF-30600-23</td>
			<td>Large-bore 3-way, male-lock, stopcocks, 10/pack, Non-sterile</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dowel Pin</td>
			<td>McMaster-Carr</td>
			<td>90145A317</td>
			<td>18-8 Stainless Steel Dowel Pins (1/32&#34; Dia. x 1/2&#34; Lg.)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Low Binding Microcentrifuge Tube</td>
			<td style="width:123px;">Corning</td>
			<td>CL S3206</td>
			<td>0.65 mL low binding snap cap microcentrifuge tube</td>
		</tr>
	</tbody>
</table>

a microfluidic platform for longitudinal imaging in caenorhabditis elegans

I det siste tiåret, microfluidic teknikker er brukt for å studere små dyr, inkludert Rundormer Caenorhabditis elegans, og har vist seg nyttig som en praktisk live bildebehandling plattform som gir muligheter for presis kontroll over eksperimentelle forhold i sanntid. I denne artikkelen viser vi live bildebehandling av personlige ansette WormSpa, en tidligere publisert egendefinert microfluidic enhet. I enheten, er flere ormer individuelt begrenset for å skille chambers, slik at multiplex langsgående overvåking av ulike biologiske prosesser. For å illustrere muligheten, utført vi bevis-av-prinsippet eksperimenter som ormer ble smittet i enheten med patogene bakterier, og dynamikken i uttrykket av immunrespons gener og egglegging var overvåkes kontinuerlig i individuelle dyr. Enkel utforming og drift av enheten gjør det egnet for brukere uten tidligere erfaring med microfluidic-baserte eksperimenter. Vi foreslår at denne tilnærmingen vil være nyttig for mange forskere interessert i langsgående observasjoner av biologiske prosesser under veldefinerte forhold.

Alder-synkroniserte ung voksen ormer (46 timer post L1 larver arrestasjonen ved 25 ° C)12 er lastet inn i enheten, som beskrevet i protokollen. Ormer lå individuelt i separate kanaler, aktivere langsgående måling av dyrene svar patogen. Når eksperimentet er vellykket, forblir de fleste ormer i deres kanaler for varigheten av eksperimentet. I dette tilfellet er bilder av personlige ormer tatt ved å plassere deres kanal i tenkelig banen slik at du slipper å fi...

Watch this Scientific Journal Video about A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans at JoVE.com

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

En Microfluidic plattform for langsgående Imaging i Caenorhabditis elegans

I denne artikkelen viser vi live bildebehandling av personlige ansette en egendefinert microfluidic enhet. I enheten, er flere ormer individuelt begrenset for å skille chambers, slik at multiplex langsgående overvåking av ulike biologiske prosesser.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

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Bioengineering

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

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Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

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A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

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Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

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Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

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An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

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Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

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Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...