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 arbeidet ble støttet av National Institutes of Health stipend (R01 HL086699, HL086699-01A2S1, 1S10RR027327-01) til MM. Vår artikkel er delvis støttet av Carl Zeiss Microimaging LLC.

Reaktive oksygen arter kan måles i levende celler ved hjelp av oksidasjon sensitive fargestoffer (1 &amp; 2) eller ved hjelp av plasmid sensorer (3 &amp; 4) ved konfokalmikroskopi. 1. Visualisering av cytosoliske ROS i J774 celler <ol><li> Grow J774.1 mus monocytt-avledet makrofager (10 6 celler / ml) på glass bunn 35 mm retter (Harvard Apparatus) i 48 h. </li><li> Challenge celler med TLR-agonister (2 mikrogram / ml, Lipo-teichoic syre-TLR2 agonist; 10μg/ml, Poly (I: C)-TLR3...

<ol>
	<li>Madesh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+role+for+superoxide+in+InsP3+receptor-mediated+mitochondrial+dysfunction+and+endothelial+apoptosis.">Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis.</a> J. Cell. Biol. 170, 1079-1090 (2005).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol. Rev. 82, 47-95 (2002).</li><li>Hamanaka, R. B., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+reactive+oxygen+species+regulate+cellular+signaling+and+dictate+biological+outcomes.">Mitochondrial reactive oxygen species regulate cellular signaling and dictate biological outcomes.</a> Trends Biochem. Sci. 35, 505-513 .</li><li>Hawkins, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-glutathionylation+activates+STIM1+and+alters+mitochondrial+homeostasis.">S-glutathionylation activates STIM1 and alters mitochondrial homeostasis.</a> J. Cell. Biol. 190, 391-405 (2010).</li><li>Madesh, M., Hajnoczky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC-dependent+permeabilization+of+the+outer+mitochondrial+membrane+by+superoxide+induces+rapid+and+massive+cytochrome+c+release.">VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release.</a> J. Cell Biol. 155, 1003-1015 (2001).</li><li>Finkel, T., Holbrook, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidants+oxidative+stress+and+the+biology+of+ageing.">Oxidants oxidative stress and the biology of ageing.</a> Nature. 408, 239-247 (2000).</li><li>Rittirsch, D., Flierl, M. A., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harmful+molecular+mechanisms+in+sepsis.">Harmful molecular mechanisms in sepsis.</a> Nat. Rev. Immunol. 8, 776-787 (2008).</li><li>Sanlioglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+induces+Rac1-dependent+reactive+oxygen+species+formation+and+coordinates+tumor+necrosis+factor-alpha+secretion+through+IKK+regulation+of+NF-kappa.">Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappa.</a> 276, 30188-30198 (2001).</li><li>Hawkins, B. J., Madesh, M., Kirkpatrick, C. J., Fisher, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+flux+in+endothelial+cells+via+the+chloride+channel-3+mediates+intracellular+signaling.">Superoxide flux in endothelial cells via the chloride channel-3 mediates intracellular signaling.</a> Mol. Biol. Cell. 18, 2002-2012 (2007).</li><li>Vliet, A. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidases+in+lung+biology+and+pathology:+host+defense+enzymes+and+more.">NADPH oxidases in lung biology and pathology: host defense enzymes and more.</a> Free Radic. Biol. Med. 44, 938-955 (2008).</li><li>Babior, B. M., Kipnes, R. S., Curnutte, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+defense+mechanisms.+The+production+by+leukocytes+of+superoxide,+a+potential+bactericidal+agent.">Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent.</a> J. Clin. Invest. 52, 741-744 (1973).</li><li>Lambeth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOX+enzymes+and+the+biology+of+reactive+oxygen.">NOX enzymes and the biology of reactive oxygen.</a> Nat. Rev. Immunol. 4, 181-189 (2004).</li><li>Mukhopadhyay, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+apoptosis+and+mitochondrial+superoxide+production+in+live+cells+by+flow+cytometry+and+confocal+microscopy.">Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy.</a> Nat. Protoc. 2, 2295-2301 (2007).</li><li>Niethammer, P., Grabher, C., Look, A. T., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-scale+gradient+of+hydrogen+peroxide+mediates+rapid+wound+detection+in+zebrafish.">A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.</a> Nature. 459, 996-999 (2009).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+innate+immunity.">Toll-like receptors and innate immunity.</a> Nat. Rev. Immunol. 1, 135-145 (2001).</li></ol>

Metoden er beskrevet her tillater rask og kvantitativ måling av reaktive oksygen arter i levende celler bruker oksidering sensitive fargestoffer eller plasmid sensorer. Agonister av TLRs (Toll-like reseptorer) er forbindelser som stimulerer cellene gjennom TLRs tilstede på celleoverflaten og utløse nedstrøms signalveier 15. I protokoll vår, brukte vi tre ulike TLR-agonister viz, Lipo-teichoic syre-TLR2 agonist, Poly (I: C).-TLR3 agonist; LPS-TLR4 agonist som er rapportert å indusere ROS som et modellsys...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalognummer </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> Endotelial vekstfaktor supplement (EKG) </td><td> Upstate </td><td> 02-102 </td></tr><tr><td> Fetal Bovine Serum </td><td> Invitrogen </td><td> 12662011 </td></tr><tr><td> G418 </td><td> Invitrogen </td><td> 10131-027 </td></tr><tr><td> Gelatin </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 Red </td><td> Invitrogen </td><td> M36008 </td></tr><tr><td> Opti-MEM jeg Redusert 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 programvare 5.0 </td><td> GraphPad Software, Inc. </td><td></td></tr><tr><td> SigmaPlot 11,0 </td><td> Systat programvare, Inc. </td><td></td></tr><tr><td> Trypsin-EDTA (10x) </td><td> Invitrogen </td><td> 15400054 </td></tr><tr><td> T-25 Flasks </td><td> Corning </td><td> 430639 </td></tr><tr><td> T-75 Flasks </td><td> BD Falcon </td><td> 353136 </td></tr><tr><td> 96-godt TC micro bra plate </td><td> BD Falcon </td><td> 353072 </td></tr><tr><td> Zen 2009 programvare </td><td> Carl Zeiss 510 Meta konfokalmikroskopi </td><td></td></tr></tbody></table>

visualization of vascular ca2+ signaling triggered by paracrine derived ros

Oksidativt stress har vært innblandet i en rekke patologiske tilstander, inkludert iskemi / reperfusjon skade og sepsis. Konseptet med oksidativt stress refererer til avvikende dannelse av ROS (reaktive oksygen arter), som inkluderer O 2 • -, H 2 O 2, og hydroksyl radikaler. Reaktive oksygen arter påvirker en rekke cellulære prosesser inkludert signaltransduksjon, celleproliferasjon og celledød 1-6. ROS har potensial til å skade vaskulære og orgel celler direkte, og kan initiere sekundære kjemiske reaksjoner og genetiske endringer som til slutt føre til en forsterkning av den første ROS-mediert vevsskade. En viktig del av forsterkningen kaskade som forverrer irreversibel vevsskade er rekruttering og aktivering av sirkulerende inflammatoriske celler. Under betennelse, inflammatoriske celler produserer cytokiner som tumor necrosis factor-α (TNF-alfa) og IL-1 somaktiverer endotelceller (EF) og epitelceller og ytterligere forsterke den inflammatoriske responsen 7. Vaskulær endotelial dysfunksjon er et etablert trekk ved akutt betennelse. Makrofager bidra til endotelial dysfunksjon ved betennelse ved mekanismer som forblir uklare. Aktivering av makrofager resultater i ekstracellulære utgivelsen av O 2 • - og ulike pro-inflammatoriske cytokiner, som utløser patologisk signalering i tilstøtende celler 8. NADPH oksidase er de viktigste og primære kilde til ROS i de fleste celletyper. Nylig er det vist av oss og andre, 9,10 som ROS produsert av NADPH oksidase indusere mitokondrie ROS produksjonen i mange patofysiologiske forhold. Derfor måle mitokondrienes ROS produksjonen er like viktig i tillegg til å måle cytosoliske ROS. Makrofager produsere ROS av flavoprotein enzymet NADPH oxidase som spiller en primær rolle i betennelse. Når aktivert,phagocytic NADPH oxidase produserer store mengder av O 2 • - som er viktige verten forsvarsmekanisme 11,12. Selv paracrine-avledet O 2 • - spiller en viktig rolle i patogenesen av vaskulære sykdommer, visualisering av paracrine ROS-indusert intracellulær signalering inkludert Ca 2 + mobilisering er fortsatt hypotesen. Vi har utviklet en modell der aktiverte makrofager blir brukt som en kilde til O 2 • - til transduce et signal til tilstøtende endotelceller. Ved hjelp av denne modellen demonstrerer vi at macrophage-avledet O 2 • - fører til kalsium signalisering i tilstøtende 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 for å få innsikt i å visualisere paracrine-avledet ROS induksjon av endotelial Ca2 + signalering er beskrevet. Denne metoden tar nytte av å måle paracrine avledet ROS utløst Ca2 + mobilisering i vaskulære endotelceller i en co-kultur modell.

Visualisering av Vaskulær Ca 2 + Signaling Utløst av parakrine Avledet 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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