Name: microfluidic-based electrotaxis for on-demand quantitative analysis of caenorhabditis elegans' locomotion
Uploaded: 2013-05-02T13:00:00
Description: Watch this Scientific Journal Video about Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion at JoVE.com

Forfatterne vil gerne takke naturvidenskab og Engineering Research Council of Canada, Canada Research Stole Program, canadiske Institutes of Health Research, og Ontario Ministeriet for forskning og innovation gennem deres Early Eliteforsker Program for finansiel støtte.

1.. Fotolitografi for Master Mold Fabrication <ol><li> Bade en 3 i. silicium wafer i acetone i 30 sekunder og derefter methanol i 30 sek. Skyl med dH 2 0 vand i 5 min. </li><li> Tør wafer overflade med en N2 slag pistol. Varm wafer på en varmeplade ved 140 ° C i 2 minutter. </li><li> Plasma oxidere overfladen af ​​silicium wafer (1 min, 50 W). </li><li> Spin-coat wafer overflade med 3 ml SU-8 100 fotoresist (40 sek 1.750 rpm). </li><li> Pre-bage det coatede wafer på en varm plade ved 65 ° C i 10 mi...

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Drage fordel af den adfærdsmæssige fænomen først beskrevet af Gabel og kolleger og bygge på dielektroforetiske manipulation arbejde Chuang og kolleger 11,12, vores microfluidic-baserede electrotaxis assay giver en nem, robust og følsom metode til at undersøge neuronal aktivitet i orme anvender bevægelse som en udgang. Analysen bevægelighed parametre muliggør kvantitativ sammenligning mellem forskellige genotyper. Præcisionen af ​​mikrokanalplade fabrikation og elektrisk felt ansøgning vedlagt ...

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microfluidic-based electrotaxis for on-demand quantitative analysis of caenorhabditis elegans' locomotion

De nematode Caenorhabditis elegans er en alsidig model organisme til biomedicinsk forskning på grund af sin bevarelse af sygdomsrelaterede gener og stier samt dets lethed til dyrkning. Adskillige C. elegans sygdomsmodeller er blevet rapporteret, herunder neurodegenerative sygdomme såsom Parkinsons sygdom (PD), hvilket indebærer degeneration af dopaminerge (DA) neuroner 1.. Både transgener og neurotoksiske kemikalier er blevet anvendt til at inducere DA neurodegeneration og deraf bevægelse defekter i orme, der giver mulighed for undersøgelser grundlag af neurodegeneration og skærme til neurobeskyttende gener og forbindelser 2,3. Skærme i lavere eukaryoter som C. elegans giver en effektiv og økonomisk middel til at identificere forbindelser og gener påvirker neuronal signalering. Konventionelle skærme er typisk udføres manuelt, og scorede ved visuel inspektion, hvorfor de er tid-consuming og tilbøjelige til menneskelige fejl. Derudover fleste fokuserer på celleniveau analyse samtidig ignorerer bevægelse, hvilket er en særlig vigtig parameter for bevægelsesforstyrrelser. Vi har udviklet en ny mikrofluid screening (figur 1), der styrer og kvantificerer C. elegans &#39;bevægelse ved hjælp af elektrisk felt stimuli inde microchannels. Vi har vist, at en jævnstrøm (DC) felt robust kan fremkalde on-demand bevægelse mod katoden (&quot;electrotaxis&quot;) 4.. Vende feltets polaritet forårsager ormen til hurtigt ændre sin retning. Vi har også vist, at defekter i dopaminerge og andre sensoriske neuroner ændrer svømning respons 5.. Derfor kan abnormaliteter i neuronal signalering bestemmes ved hjælp af bevægelse som en read-out. Bevægelsen respons kan præcist kvantificeres ved hjælp af en række parametre, såsom svømning hastighed, krop bøjning frekvens og tilbageførsel tid. <p class = &quot;jove_content&quot;&gt; Vores arbejde har afsløret, at den electrotactic respons varierer med alderen. Konkret unge voksne reagere på et lavere udbud af elektriske felter og bevæge sig hurtigere i forhold til larver 4.. Disse fund førte os til at designe en ny mikrofluid enhed til passivt at sortere orme efter alder og fænotype 6.. Vi har også testet reaktion orme til pulserende DC og vekselstrøm (AC) elektriske felter. Impuls DC marker med forskellige driftscyklusser effektivt genererede electrotaxis i både C. elegans og dens fætter C. briggsae 7.. I et andet eksperiment, immobiliseret symmetriske AC felter med frekvenser fra 1 Hz til 3 KHz orme inde i kanalen 8. Gennemførelsen af ​​det elektriske felt i en mikrofluid miljø giver mulighed for hurtig og automatiseret udførelse af electrotaxis analysen. Denne fremgangsmåde lover at lette high-throughput genetiske og kemiske skærme til faktorerpåvirker neuronal funktion og levedygtighed.

En repræsentativ video af en vildtype unge voksne nematode s electrotaxis og dets position og hastighed udgange fra ormen tracking software er vist i Supplerende Video 1 og figur 3. Bevægelsen analyse software i sig selv kan ikke genkende retningen af ​​marken polaritet og tidspunktet for polaritetsreversering, men snarere, skal disse oplysninger indhentes fra kilden video. Dette kunne gøres ved hjælp af en lyd-eller visuel i videoen eller skrive ned eksperimentelle betingelser ...

Watch this Scientific Journal Video about Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion at JoVE.com

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

En semi-automatiseret MikroElektroMekaniske strømningsteknisk metode til at inducere on-demand bevægelse i<em&gt; Caenorhabditis elegans</em&gt; Beskrives. Denne metode er baseret på den neurofysiologiske fænomenet orme reagerer på milde elektriske felter (&quot;electrotaxis&quot;) inden mikrofluidkanaler. Mikrofluid electrotaxis fungerer som en hurtig, følsom, billig og skalerbar teknik til at screene for faktorer, der påvirker neuronal sundhed.

Mikrofluid-baserede Electrotaxis for On-demand kvantitativ analyse af<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

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Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

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