Name: determination of lipid raft partitioning of fluorescently-tagged probes in living cells by fluorescence correlation spectroscopy (fcs)
Uploaded: 2012-04-06T13:00:00
Description: Watch this Scientific Journal Video about Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS at JoVE.com

Dette arbeidet ble støttet av en bevilgning fra Agence Nationale de la Recherche (ChoAD). Vi er også takknemlige til Fondation ICM (Institut du Cerveau et de la Moelle) for deres økonomiske støtte.

1. Kalibrering av FCS Setup <ol><li> Start confocal mikroskop, lasere, datamaskiner, inkubator for temperatur og CO 2 kontroll. </li><li> Kontroller at SPAD (Single Photon Avalanche Diode) er på og fluorescens filteret inne i SPAD er godt egnet til prøven din. Kontroller at SPAD er synkronisert i tid. Beware å bare starte FCS programvare så snart dine SPAD innstillingene er klare for oppkjøp. </li><li> Forbered en frisk løsning av koleratoksin-Alexa488 fortynnet i PBS å nå en konsentrasjon på 1 mg ...

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Den FCS metoden presenteres her gir en sensitiv og rask analyse av lipid flåten partisjonering av fluorescerende prober interessante i levende celler. FCS kombinerer nøyaktigheten av lokalisering av konfokalmikroskopi med følsomheten enkelt foton telling. Den største forskjellen mellom FCS og standard biokjemiske teknikker er at FCS gjør den absolutte bestemmelse av lipid flåter partisjonen av målet og ikke relative partisjonen som er tilfellet for DRMS ​​isolasjon eller co-patching. <p class="jove_conten...

<html><table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Koleratoksin subenhet B-Alexa 488 </td><td> Invitrogen </td><td> C-34775 </td><td> MW (pentamer) = 57 kg / mol </td></tr><tr><td> Confocal mikroskop </td><td> Leica </td><td> SP5 </td><td></td></tr><tr><td> Inkubator for temperatur og CO 2 kontroll </td><td> Livet bildetjenester </td><td> The Cube og Box </td><td></td></tr><tr><td> SPAD (Single Photon Avalanche Diode) </td><td> MPD (Micro Photon Devices) </td><td> PDM serie (100 mikrometer følsomt område) </td><td></td></tr><tr><td> Høy pass 488 nm filter </td><td> Semrock </td><td> 488 nm blokkerende kant BrightLine lang-pass filter Part #ails.aspx? id = FF01-488/LP-25 &quot;target =&quot; _blank &quot;&gt; FF01-488/LP-25 </td><td></td></tr><tr><td> FCS deteksjon enhet </td><td> Picoquant </td><td> Picoharp 300 modul </td><td></td></tr><tr><td> Erverv og auto-korrelasjon programvare </td><td> Picoquant </td><td> SymPhoTime </td><td></td></tr><tr><td> Montering programvare </td><td> OriginLab </td><td> OriginPro8 </td><td></td></tr></tbody></table>

determination of lipid raft partitioning of fluorescently-tagged probes in living cells by fluorescence correlation spectroscopy (fcs)

I de siste femten årene den oppfatningen at cellemembraner er ikke homogent og stole på microdomains å utøve sine funksjoner er blitt allment akseptert. Lipid flåter er membran microdomains anriket på kolesterol og sphingolipids. De spiller en rolle i cellulære fysiologiske prosesser som signalering og menneskehandel 1,2, men er også antatt å være sentrale aktører i flere sykdommer, inkludert virus-eller bakterieinfeksjoner og nevrodegenerative sykdommer 3. Likevel deres eksistens er fortsatt et spørsmål om uenighet 4,5. Faktisk har lipid flåte størrelsen er anslått å være rundt 20 nm 6, langt under oppløsningen grensen av konvensjonell mikroskopi (rundt 200 NM), og dermed utelukker deres direkte avbildning. Inntil nå, var de viktigste teknikkene som brukes til å vurdere delingen av proteiner av interesse inne lipid flåter Vaskemiddel Resistente membraner (DRMS) isolasjon og co-patching med antistoffer. Selv om mye brukt becabruk av deres ganske enkel implementering, var disse teknikkene tilbøyelige til gjenstander og dermed kritisert 7,8. Tekniske forbedringer var derfor nødvendig å overvinne disse gjenstandene, og for å kunne undersøke lipid flåter partisjon i levende celler. Her presenterer vi en metode for sensitiv analyse av lipid flåtene partisjon av fluorescently-merkede proteiner eller lipider i plasmamembranen av levende celler. Denne metoden kalles fluorescens korrelasjon spektroskopi (FCS), avhengig av ulikheten i diffusjon tider med fluorescerende prober plassert innsiden eller utsiden av lipid flåter. Faktisk, noe som gjenspeiles i både kunstig membraner og cellekulturer, ville sonder spre mye raskere ute enn inne tette lipid flåter 9,10. For å bestemme diffusjon ganger, er liten fluorescens svingninger målt som en funksjon av tid i en fokal volum (ca. 1 femtoliter), som ligger ved plasma membran av celler med en confocal mikroskop (Fig.1). De auto-korrelasjon kurver kan da trekkes fra disse svingningene og utstyrt med egnede matematiske spredning modeller 11. FCS kan brukes til å bestemme lipid flåten oppdeling av ulike sonder, så lenge de er fluorescently tagget. Fluoriserende tagging kan oppnås ved uttrykk av fluorescerende fusion proteiner eller ved binding av fluorescerende ligander. Videre kan FCS brukes ikke bare i kunstige membraner og cellelinjer, men også i grunnskolen kulturer, som beskrevet nylig tolv. Den kan også brukes til å følge dynamikken i lipid flåte partisjonering etter narkotika tillegg eller membran lipidsammensetning forandring 12.

Watch this Scientific Journal Video about Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS at JoVE.com

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

En teknikk for å sondere lipid flåten partisjonering av fluorescerende proteiner på plasmamembranen av levende celler er beskrevet. Det tar nytte av misforhold i diffusjon tider med proteiner lokalisert innenfor eller utenfor lipid flåter. Oppkjøp kan utføres dynamisk i kontroll forhold eller etter narkotika tillegg.

Bestemmelse av Lipid Raft Partisjonering av fluorescently-merkede prober i levende celler ved fluorescens korrelasjon spektroskopi (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

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Biology

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

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