Name: measurement of cellular chemotaxis with ecis/taxis
Uploaded: 2012-04-01T11:00:00
Description: Watch this Scientific Journal Video about Measurement of Cellular Chemotaxis with ECIS/Taxis at JoVE.com

Dette arbejde blev støttet af tilskud fra National Institutes of Health (ES07408 og EB00208).

1. ECIS / Taxa Electrode Forberedelse <ol><li> Guld overflade ECIS / taxaer elektrodesæt (bestående af 8 kamre pr objektglas) først stabiliseres ved forbehandling med steril 10 mM cystein i deioniseret vand (dH2O) i 15 minutter ved stuetemperatur under sterile betingelser. </li><li> Aspirer cystein opløsning fra hver elektrode kammer skylles 3 gange med sterilt dH2O, og erstat med 250 ul komplet medium (RPMI 1640, 10% FBS, 25 mM HEPES-puffer). </li><li> Slut elektrodeopstillingen at kontakte ...

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Nye egenskaber ECIS / Taxis analysen omfatter dens evne til at automatisere indsamling af data i realtid, som cellerne reagerer på chemoattraktant. Mens den mest almindelige anvendelse af denne teknologi er at måle cellulære responser til individuelle kemotaktiske gradienter eller gradienter sammensat af blandinger af kemotaksi agonister og antagonister, til ECIS / taxier fremgangsmåde er også egnet til variationer af disse konfigurationer, som kan være ganske nyttige i Vurderingen af ​​cellulær respons. Der ...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalog nr. </td><td> Kommentarer </td></tr><tr><td> ECIS Zθ </td><td> Anvendt Biofysik </td><td></td><td> <a href="http://www.biophysics.com/prodducts_Ecisz0.php">http://www.biophysics.com/prodducts_Ecisz0.php</a> </td></tr><tr><td> ECIS elektrodeformation </td><td> Anvendt Biofysik </td><td> 8W Kemotaxi </td><td> <a href="http://www.biophysics.com/cultureware.php">http://www.biophysics.com/cultureware.php</a> </td></tr><tr><td> SeaKem GTG agarose </td><td> BioWhittaker Molekylære Applikationer </td><td> 50070 </td><td></td></tr><tr><td> RPMI1640 </td><td> Cellgro </td><td> 10-040 </td><td></td></tr><tr><td> HyClone føtalt bovint serum </td><td> Thermo Scientific </td><td> SH300703 </td><td></td></tr><tr><td> Penicillin / Streptomycin </td><td> MP Biomedicals </td><td> 1670049 </td><td> Penicillin 5000 IU / ml; Streptomycin 5 mg / ml </td></tr><tr><td> HEPES puffer </td><td> MP Biomedicals </td><td> 1688449 </td><td> 1 M opløsning, cellekultur grad </td></tr><tr><td> 14 gauge rustfri stålkanyle (2) 4 inch </td><td> General Laboratory Supply </td><td> 5-8365-1 </td><td> Blunt punkt </td></tr></tbody></table>

measurement of cellular chemotaxis with ecis/taxis

Cellulære bevægelse som reaktion på ydre stimuli er afgørende for mange cellulære processer, herunder sårheling, inflammation og respons på infektion. En almindelig metode til at måle kemotaksi er Boyden kammer assay, i hvilket celler og kemotiltrækkende er adskilt af en porøs membran. Som celler migrerer gennem membranen mod det kemotiltrækkende de klæber til undersiden af membranen eller kan opdeles i de underliggende mediet, og efterfølgende farvet og visuelt tælles 1. Ved denne fremgangsmåde celler eksponeres for en stejl og transient kemoattraktant gradient, hvilket menes at være en dårlig gengivelse af gradienter, der findes i væv to. Et andet assaysystem, under-agarose-kemotaxi-assay, 3, 4 foranstaltninger cellebevægelse over et fast substrat med en tynd vandig film, der dannes under agarose lag. Den gradient, der udvikler i agarose er lavvandet, og menes at være en appropriate repræsentation af naturligt forekommende gradienter. Kemotaksi kan vurderes ved mikroskopisk afbildning af den tilbagelagte afstand. Både Boyden-kammer-assayet og under-agarose assay sædvanligvis udformet som slutpunkt assays. Den automatiserede ECIS / Taxa system kombinerer under-agarose tilgang med Electric Cell-substrat Impedans Sensing (ECIS) 5, 6. I dette assay, er target elektroder placeret i hver af 8 kamre. En stor modelektrode løber gennem hver af de 8 kamre (figur 2). Hvert kammer er fyldt med agarose og to små brønde er snit i agarosen på hver side af målet elektroden. En brønd fyldes med prøven cellepopulation, medens den anden holder kilder spredende kemoattraktant (figur 3). Strøm ledes gennem systemet kan anvendes til at bestemme ændringen i modstand, der opstår som celler passerer hen over mål-elektroden. Celler på målet electrode forøge modstandsdygtigheden af systemet 6. Desuden udgør hurtige fluktuationer i modstanden ændringer i interaktioner af celler med elektrodeoverfladen og er indikative for løbende cellulære formændringer. Den ECIS / Taxis system kan måle bevægelsen af ​​cellepopulationen i real-tid over længere tid, men er også følsom nok til at detektere ankomsten af ​​en enkelt celle ved målet elektroden. Dictyostelium discoidium vides at migrere i nærvær af en folat gradient 7, 8 og kemotaktiske respons kan måles nøjagtigt ved ECIS / Taxis 9. Leukocytkemotaxis, har som reaktion på SDF1α og kemotaxi antagonister også blevet målt med ECIS / Taxis 10, 11. Et eksempel på leukocyt respons på SDF1α er vist i fig 1.

Watch this Scientific Journal Video about Measurement of Cellular Chemotaxis with ECIS/Taxis at JoVE.com

Measurement of Cellular Chemotaxis with ECIS/Taxis

Den ECIS / Taxa system er en automatiseret, real-time analyse, der måler cellulære kemotaksi. I dette assay flytte cellerne under et lag af agarose at nå frem til et mål elektrode. Cellulær bevægelse måles ved indtræden af ​​resistens over for vekselstrøm 0.

Måling af cellulære Kemotaksi med ECIS / Taxa

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

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