Name: visualization of vascular ca2+ signaling triggered by paracrine derived ros
Uploaded: 2011-12-21T13:00:00
Description: Watch this Scientific Journal Video about Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS at JoVE.com

Dette arbejde blev støttet af National Institutes of Health tilskud (R01 HL086699, HL086699-01A2S1, 1S10RR027327-01) til MM. Vores artikel er delvis støttet af Carl Zeiss Microimaging LLC.

Reaktive ilt arter kan måles i levende celler ved hjælp af oxidation følsomme farvestoffer (1 &amp; 2) eller ved anvendelse af plasmidet sensorer (3 &amp; 4) af konfokal mikroskopi. 1. Visualisering af cytosole ROS i J774 celler <ol><li> Grow J774.1 musen monocyt-afledte makrofager (10 6 celler / ml) på glasbund 35-mm retter (Harvard apparater) i 48 h. </li><li> Challenge celler med TLR agonister (2 mg / ml, Lipo-teichoic syre-TLR2 agonist, 10μg/ml, Poly (I: C)-TLR3 agonist...

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Metoden beskrives her giver hurtig og kvantitativ måling af reaktive ilt arter i levende celler, enten ved hjælp af oxidation følsomme farvestoffer eller plasmid sensorer. Agonister af TLRs (Toll-lignende receptorer) er stoffer, der stimulerer celler gennem TLRs stede på celleoverfladen og udløse downstream signalveje 15. I vores protokol, brugte vi tre forskellige TLR agonister nemlig, Lipo-teichoic syre-TLR2 agonist, Poly (I: C).-TLR3 agonist; LPS-TLR4 agonist, som er rapporteret at inducere ROS som et...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Attofluor celle kammer </td><td> Invitrogen </td><td> A7816 </td></tr><tr><td> Antimycin A </td><td> Sigma Aldrich </td><td> A8674 </td></tr><tr><td> DMEM lav glukose medium </td><td> Invitrogen </td><td> 10567-014 </td></tr><tr><td> Endotel vækstfaktor supplement (EKG) </td><td> Upstate </td><td> 02-102 </td></tr><tr><td> Føtalt bovint serum </td><td> Invitrogen </td><td> 12662011 </td></tr><tr><td> G418 </td><td> Invitrogen </td><td> 10131-027 </td></tr><tr><td> Gelatine </td><td> Sigma Aldrich </td><td> G1393 </td></tr><tr><td> H 2 DCFDA </td><td> Invitrogen </td><td> D-399 </td></tr><tr><td> LPS </td><td> Sigma Aldrich </td><td> L4516 </td></tr><tr><td> LTA </td><td> Sigma Aldrich </td><td> L2515 </td></tr><tr><td> MitoSOX Rød </td><td> Invitrogen </td><td> M36008 </td></tr><tr><td> Opti-MEM jeg Reduceret Serum Medium </td><td> Invitrogen </td><td> 51985091 </td></tr><tr><td> Pen / Strep (10x) </td><td> Invitrogen </td><td> 15140163 </td></tr><tr><td> pHyPer-CYTO </td><td> Evrogen </td><td> FP941 </td></tr><tr><td> pHyPer-dMito </td><td> Evrogen </td><td> FP942 </td></tr><tr><td> Poly (I: C) </td><td> Sigma Aldrich </td><td> P0913 </td></tr><tr><td> Prism Software 5.0 </td><td> GraphPad Software, Inc. </td><td></td></tr><tr><td> SigmaPlot 11,0 </td><td> Systat software, Inc. </td><td></td></tr><tr><td> Trypsin-EDTA (10x) </td><td> Invitrogen </td><td> 15400054 </td></tr><tr><td> T-25 Kolber </td><td> Corning </td><td> 430639 </td></tr><tr><td> T-75 Kolber </td><td> BD Falcon </td><td> 353136 </td></tr><tr><td> 96-såvel TC mikro brønds plade </td><td> BD Falcon </td><td> 353072 </td></tr><tr><td> Zen 2009 software </td><td> Carl Zeiss 510 Meta konfokal mikroskopi </td><td></td></tr></tbody></table>

visualization of vascular ca2+ signaling triggered by paracrine derived ros

Oxidativt stress har været impliceret i en række patologiske tilstande herunder iskæmi / reperfusion skader og sepsis. Begrebet oxidative stress refererer til afvigende dannelse af ROS (reaktive ilt arter), som omfatter O 2 • -, H 2 O 2, og hydroxyl radikaler. Reaktive ilt arter påvirker en lang række cellulære processer, herunder signaltransduktion, celledeling og celledød 1-6. ROS har potentiale til at skade kar og orgel celler direkte, og kan iværksætte sekundære kemiske reaktioner og genetiske forandringer, der i sidste ende resulterer i en forstærkning af den oprindelige ROS-medieret vævsskader. Et centralt element i den forstærkning kaskade, der forværrer irreversibel vævsskade er rekruttering og aktivering af cirkulerende inflammatoriske celler. I løbet af inflammation, producerer inflammatoriske celler cytokiner såsom tumor nekrose faktor-α (TNF) og IL-1, deraktiverer endotelceller (EF) og epitelceller og yderligere øge det inflammatoriske respons 7. Vaskulær endotel dysfunktion er et fast indslag af akut inflammation. Makrofager bidrage til endotel dysfunktion i løbet af inflammation af mekanismer, som er fortsat uklare. Aktivering af makrofager resultater i den ekstracellulære frigivelse af O 2 • - og forskellige pro-inflammatoriske cytokiner, som udløser patologiske signalering i tilstødende celler 8. NADPH oxidaser er den største og primære kilde til ROS i de fleste celletyper. For nylig er det vist ved os og andre, 9,10, at ROS produceres af NADPH oxidaser inducere mitokondrie ROS produktion i mange patofysiologiske forhold. Derfor måle mitokondrier ROS produktion er lige så vigtigt som udover at måle cytosolisk ROS. Makrofager producerer ROS af flavoprotein enzymet NADPH oxidase, som spiller en primær rolle i inflammation. Når den er aktiveret,fagocyterende NADPH oxidase producerer rigelige mængder af O 2 • - der er vigtige i værtslandet forsvar mekanisme 11,12. Selvom paracrine-afledte O 2 • - spiller en vigtig rolle i patogenesen af kar-sygdomme, visualisering af paracrine ROS-induceret intracellulære signaler, herunder Ca 2 + mobilisering er stadig hypotese. Vi har udviklet en model, hvor aktiverede makrofager bruges som en kilde til O 2 • - at transduce et signal til tilstødende endotelceller. Ved hjælp af denne model vi vise, at makrofag-afledt O 2 • - føre til calcium signalering i de tilstødende endotelceller.

Watch this Scientific Journal Video about Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS at JoVE.com

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

En effektiv metode til at få indblik i at visualisere paracrine-afledte ROS induktion af endothel Ca2 + signalering er beskrevet. Denne metode benytter sig af at måle paracrine afledt ROS udløst Ca2 + mobilisering i vaskulære endotelceller i en co-kultur model.

Visualisering af Vascular Ca 2 + Signaling Triggered ved Paracrine Afledte ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

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Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

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