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 arbejde blev støttet af en bevilling fra Agence Nationale de la Recherche (ChoAD). Vi er også taknemmelige for den Fondation ICM (Institut du Cerveau et de la Moelle) for deres økonomiske støtte.

1. Kalibrering af FCS opsætning <ol><li> Start konfokal mikroskop, lasere, computere, inkubator for temperatur og CO 2 kontrol. </li><li> Sørg for, at SPAD (Single Photon Avalanche Diode) er tændt, og fluorescens filteret i SPAD er velegnet til din prøve. Kontroller, at SPAD er synkroniseret i tid. Pas på kun at starte din FCS-softwaren, når dine SPAD indstillinger er klar til overtagelse. </li><li> Fremstilling af en frisk opløsning af koleratoksin-Alexa488 fortyndet i PBS for at opnå en koncentrat...

<ol>
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FCS Fremgangsmåden præsenteret her muliggør en følsom og hurtig analyse af lipidklump opdelingen af ​​fluorescerende prober af interesse i levende celler. FCS kombinerer nøjagtigheden af ​​placeringen af ​​konfokal mikroskopi med følsomheden af ​​en enkelt foton-tælling. Den største forskel mellem FCS og standard biokemiske teknikker er, at FCS muliggør absolut bestemmelse af lipidklumper deling af målet og ikke den relative partition som det er tilfældet for DRM isolering eller co-lapning. </...

<html><table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Koleratoxin underenhed B-Alexa 488 </td><td> Invitrogen </td><td> C-34.775 </td><td> MW (pentamer) = 57 kg / mol </td></tr><tr><td> Konfokalt mikroskop </td><td> Leica </td><td> SP5 </td><td></td></tr><tr><td> Inkubator for temperatur og CO 2-kontrol </td><td> Livet billedbehandling tjenester </td><td> Det Cube og the Box </td><td></td></tr><tr><td> SPAD (Single Photon Avalanche Diode) </td><td> MPD (Micro Photon Devices) </td><td> PDM serie (100 um følsomt område) </td><td></td></tr><tr><td> Højpas 488 nm filter </td><td> Semrock </td><td> 488 nm blokerende kant BrightLine langpasfilter 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 detektionsenheden </td><td> Picoquant </td><td> Picoharp 300 modul </td><td></td></tr><tr><td> Køb og auto-korrelation software </td><td> Picoquant </td><td> SymPhoTime </td><td></td></tr><tr><td> Tilpasningssoftware </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 sidste femten år den opfattelse, at cellemembraner ikke er homogene og stole på mikrodomæner at udøve deres funktioner er blevet bredt accepteret. Lipidklumper er membran mikrodomæner beriget med cholesterol og sphingolipider. De spiller en rolle i cellulære fysiologiske processer, såsom signalering, og handel 1,2, men er også menes at være centrale aktører i en række sygdomme, herunder virale eller bakterielle infektioner og neurodegenerative sygdomme 3. Men deres eksistens er stadig et spørgsmål om uenighed 4,5. Faktisk har lipidklump størrelse er anslået til at være omkring 20 nm 6, langt under opløsning grænse for konventionel mikroskopi (ca. 200 nm), således til hinder for deres direkte billeddannelse. Indtil nu, var de vigtigste teknikker, der anvendes til at vurdere delingen af ​​proteiner af interesse inde lipidklumperne Vaskemiddel Resistant Membraner (DRM) isolation og co-lappe med antistoffer. Selvom udbredt becabrug af deres ret let gennemføres, disse teknikker var tilbøjelige til artefakter og dermed kritiseret 7,8. Tekniske forbedringer var derfor nødvendigt at overvinde disse artefakter og at være i stand til at undersøge lipidklumper skillevæg i levende celler. Her præsenterer en fremgangsmåde til følsom analyse af lipidklumper delingen af ​​fluorescens-mærkede proteiner eller lipider i plasmamembranen af ​​levende celler. Denne fremgangsmåde betegnes fluorescenskorrelationsspektroskopi (FCS), baseret på forskellene i udbredelse tider fluorescerende sonder eller udenfor lipidklumper. Faktisk, som det ses i både kunstige membraner og cellekulturer vil proberne diffundere meget hurtigere uden end indersiden tætte lipidklumper 9,10. At bestemme diffusion gange, minutters fluorescens udsving målt som en funktion af tid i en fokal volumen (ca. 1 femtoliter), placeret på plasmamembranen af celler med et konfokalt mikroskop (fig.1). Autokorrelationen kurver kan derefter trækkes af disse udsving og forsynet med passende matematiske diffusionsmodeller 11. FCS kan anvendes til at bestemme lipidklump opdeling af forskellige prober, så længe de fluorescerende er kodet. Fluorescerende mærkning kan opnås ved ekspression af fluorescerende fusionsproteiner eller ved binding af fluorescerende ligander. Endvidere kan FCS kan ikke blot anvendes i kunstige membraner og cellelinier, men også i primære kulturer, som beskrevet for nylig 12. Det kan også anvendes til at følge dynamik lipidklump separation efter lægemiddelindgivelse tilsætning eller membran lipidsammensætningen ændring 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 teknik til at undersøge lipidklump opdelingen af ​​fluorescerende proteiner til plasmamembranen af ​​levende celler er beskrevet. Den udnytter forskellen i diffusionstider for proteiner i eller uden for lipidklumper. Erhvervelse kan udføres dynamisk i kontrol betingelser eller efter narkotika tilsætning.

Bestemmelse af lipidklump opdeling af fluorescens-mærkede prober i levende celler ved fluorescenskorrelationsspektroskopi (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)

Measuring the Kinetics of mRNA Transcription in Single Living Cells

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Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

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Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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Fluorescensimagografi med One-nanometer Nøjagtighed (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

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Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

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Et grafisk brugergrænseflade til softwareassisteret sporing af proteinkoncentration i dynamiske cellulære protrusioner

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

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Konfokal mikroskopi afslører celle overflade Receptor sammenlægning gennem billedet korrelation spektroskopi

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

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Bestemmelse af Plasma membran partitionering for perifert forbundet proteiner

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

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Oligomerization dynamik af celleoverflade receptorer i levende celler ved total intern refleksion Fluorescens mikroskopi kombineret med tal og lysstyrke analyse

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Sondering mRNA kinetik i rum og tid i Escherichia coli ved hjælp af to-color single-molecule fluorescens In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...