Name: isolation of functional cardiac immune cells
Uploaded: 2011-12-05T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Dette arbejde blev støttet af en UNCF-Merck Graduate Science Research Dissertation Fellowship (JLM), National Heart, Lung, og Blood Institute giver RO1-HL-62228 (JSJ) og RO1-HL-073990 (JSJ).

1. Udarbejdelse af Hanks &#39;Balance Salt Solution (HBSS) <ol><li> Autoklave 1 L glas bægerglas, dimitteret 1000 mL cylinder, og rør bar. </li><li> Opløs HBSS pulveret løsning i autoklaveres 1 L glas bægerglas til 900 mL mærket med dobbeltdestilleret vand. </li><li> Bland HBSS løsning med 3,4 g HEPES Na + salt og pH indstilles til en værdi mellem 7,35-7,41 enten ved hjælp af koncentreret HCl eller NaOH. </li><li> Når den ønskede pH-værdien er nået, og forbliver stabil, tilsættes 5 g af bovin serum album...

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	<li>Brower, G. L., Chancey, A. L., Thanigaraj, S., Matsubara, B. B., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+and+effect+relationship+between+myocardial+mast+cell+number+and+matrix+metalloproteinase+activity.">Cause and effect relationship between myocardial mast cell number and matrix metalloproteinase activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 283, H518-H525 (2002).</li><li>Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+inhibition+of+mast+cell+degranulation+prevents+left+ventricular+remodeling+induced+by+chronic+volume+overload+in+rats.">Pharmacologic inhibition of mast cell degranulation prevents left ventricular remodeling induced by chronic volume overload in rats.</a> Journal of Cardiac Failure. 11, 548-556 (2005).</li><li>Chancey, A. L., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mast+cell-mediated+activation+of+gelatinase+and+alteration+of+ventricular+diastolic+function.">Cardiac mast cell-mediated activation of gelatinase and alteration of ventricular diastolic function.</a> Am. J. Physiol. Heart. Circ. Physiol. 282, H2152-H2158 (2002).</li><li>Levick, S. P., Gardner, J. D., Holland, M., Hauer-Jensen, M., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+adverse+myocardial+remodeling+secondary+to+chronic+volume+overload+in+mast+cell+deficient+rats.">Protection from adverse myocardial remodeling secondary to chronic volume overload in mast cell deficient rats.</a> Journal of Molecular and Cellular Cardiology. 43, 56-61 (2008).</li><li>Levick, S. P., McLarty, J. L., Murray, D. B., Freeman, R. M., Carver, W. E., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Mast+Cells+Mediate+Left+Ventricular+Fibrosis+in+the+Hypertensive+Rat.">Cardiac Mast Cells Mediate Left Ventricular Fibrosis in the Hypertensive Rat.</a> Hypertension. 53, 1041-1047 (2009).</li><li>Batista, M. L., Santos, R. V., Cunha, L. M., Mattos, K., Oliveira, E. M., Seelander, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+pro-inflammatory+cytokine+production+and+peritoneal+macrophage+function+in+rats+with+chronic+heart+failure.">Changes in the pro-inflammatory cytokine production and peritoneal macrophage function in rats with chronic heart failure.</a> Cytokine. 34, 284-290 (2006).</li><li>Galli, S. J., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+Cells:+Versatile+regulator+of+inflammation,+tissue+remodeling,+host+defense+and+homeostasis.">Mast Cells: Versatile regulator of inflammation, tissue remodeling, host defense and homeostasis.</a> Journal of Dermatological Science. 49, 7-19 (2008).</li><li>Yndestad, A., Holm, A. M., Muller, F., Simonsen, S., Froland, S. S., Gullested, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+of+inflammatory+cytokines+and+activation+markers+in+T-cells+from+patients+with+chronic+heart+failure.">Enhanced expression of inflammatory cytokines and activation markers in T-cells from patients with chronic heart failure.</a> Cardiovasc. Res. 60, 141-146 (2003).</li><li>Bozkurt, B., Kribbs, S. B., Clubb, F. J., Michael, L. H., Didenko, V. V., Hornsby, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+Relevant+Concentrations+of+Tumor+Necrosis+Factor-&#x0251;+Promote+Progressive+Left+Ventricular+Dysfunction+and+Remodeling+in+Rats.">Pathophysiologically Relevant Concentrations of Tumor Necrosis Factor-&#x0251; Promote Progressive Left Ventricular Dysfunction and Remodeling in Rats.</a> Circulation. 97, 1382-1391 (1998).</li><li>Frangogiannis, N. G., Lindsey, M. L., Michael, L. H., Youker, K. A., Bressler, R. B., Mendoza, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Cardiac+Mast+Cells+Degranulate+and+Release+Preformed+TNF-&#x0251;,+Initiating+the+Cytokine+Cascade+in+Experimental+Canine+Myocardial+Ischemia/Reperfusion.">Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-&#x0251;, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion.</a> Circulation. 98, 699-710 (1998).</li><li>Jobe, L. J., Melendez, G. C., Levick, S. P., Du, Y., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-&#945;+inhibition+attenuates+adverse+myocardial+remodeling+in+a+rat+model+of+volume+overload.">TNF-&#945; inhibition attenuates adverse myocardial remodeling in a rat model of volume overload.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, H1462-H1468 (2009).</li><li>Forman, M. F., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+histamine+secretion+during+isolation+of+rat+cardiac+mast+cells.">Spontaneous histamine secretion during isolation of rat cardiac mast cells.</a> Inflammation Research. 53, 453-457 (2004).</li><li>Levick, S. P., Murray, D. B., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+nervous+system+modulation+of+inflammation+and+remodeling+in+the+hypertensive+heart.">Sympathetic nervous system modulation of inflammation and remodeling in the hypertensive heart.</a> Hypertension. 55, 270-276 (2010).</li><li>Morgan, L., Levick, S., Voloshenyuk, T., Murray, D., Forman, M., Brower, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+isolating+functional+mast+cells+from+the+heart.">A novel technique for isolating functional mast cells from the heart.</a> Inflammation Research. 57, 241-246 (2008).</li></ol>

Denne metode til at isolere hjerte immunceller muliggør en analyse af deres funktion, fænotype, og modenhed uden uønskede aktivering, der typisk opstår under en traditionel enzymatisk fordøjelse isolation teknik. Den gennemsnitlige samlede antal celler, der opnås med denne teknik er omkring 400.000 per rotte hjerte, og forskellen farvning og flowcytometri analyse af disse celler indikerer blandet befolkning af T-lymfocytter (~ 70%), makrofager (~ 12%) og mast celler med brugen af denne teknik. (~ 12%). 13, 14...

<table border="1"><tbody><tr><td> Navn på Reagens og udstyr </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Hanks &#39;Balanced Salt </td><td> Sigma </td><td> H4891 </td><td> En flaske gør 1 L af Hanks &#39;Balance Salt Solution. Opløs Hanks &#39;Balance Salt Løsning som en del af proceduren. </td></tr><tr><td> Antibiotika-Antimyotic </td><td> Gibco </td><td> 15240-062 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3784 </td><td></td></tr><tr><td> Bovine Serum Albumin </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> 250 mL Nalogene Filter Bottle </td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N HCL </td><td> Fisher Scientific </td><td> CAS 7647-01-0 </td><td> For 6 N, tilsættes lige store mængder af HCL til deioniseretvand. </td></tr><tr><td> 10 N NaOH </td><td> Fisher Scientific </td><td> SC267 </td><td> For 5 N, tilsættes lige store mængder af NaOH i deioniseret vand. </td></tr><tr><td> Dobbeltdestilleret vand </td><td></td><td></td><td></td></tr></tbody></table> Tabel over særligt udstyr: <table border="1"><tbody><tr><td> Navn på Reagens og udstyr </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> 3-O sutur Silk Flettet </td><td> Ethicon Produkter </td><td> SA64H </td><td></td></tr><tr><td> Small Animal Ventilator </td><td> CWE Incorporated </td><td> SAR-830 </td><td> Indstil vejrtrækning til 40,5 vejrtrækninger pr min. </td></tr><tr><td> Bevel 18 gauge kanyle </td><td> BD Biosciences </td><td> 305199 </td><td> Trim nål med 1 / 2 til 3 / 4 af sin oprindelige længde til brug som en trakeale rør. </td></tr><tr><td> Teflon spids (med nål fjernet) af 24-gauge Baxter quick-Cath kateter </td><td> Baxter </td><td> 2N111 </td><td> Reducer Teflon Tip længden af ​​1 / 2 til 3 / 4. </td></tr><tr><td> 10 ml sprøjte </td><td> Fisher Scientific </td><td> 14 til 823-16B </td><td></td></tr><tr><td> 15 ml Falcon rør </td><td> BD Biosciences </td><td> 352097 </td><td></td></tr><tr><td> Sorvall RT6000B Køle-Centrifuge </td><td> Fisher Scientific </td><td> 75-004-377 </td><td> Husk at pre-cool og holde ved 4 ° C. </td></tr><tr><td> Hyclone </td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td> Hemocytometer </td><td> Hausser Videnskabelige </td><td> HS-LB-1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

Cardiac immunceller er stigende interesse for de roller, de spiller i den patologiske remodeling i mange hjertesygdomme 1-5 Disse immunceller, som omfatter mast celler, T-celler og makrofager;. Opbevare og frigive en række af biologisk aktive mediatorer, herunder cytokiner og proteaser, såsom tryptase. 6-8 Disse mæglere har vist sig at være centrale aktører i ekstracellulære matrix stofskifte ved at aktivere matrixmetalloproteinaser eller forårsager kollagen ophobning ved at modulere hjertefunktionen fibroblaster &#39;funktionen. 9-11 dog tilgængelige teknik til at isolere hjerte immunceller har været problematisk, fordi de bruger bakteriel collagenase at fordøje myokardiets væv. Denne teknik medfører aktivering af immunceller og dermed et tab af funktion. For eksempel bliver hjertets mastceller betydeligt mindre lydhøre over for stoffer, der forårsager degranulering. 12 Derfor har vi udviklet en teknik, der giver mulighed for than isolering af funktionelle hjerte immunceller, som ville føre til en bedre forståelse af den rolle, disse celler i hjertesygdom. 13, 14 Denne metode kræver et kendskab til de anatomiske placering af rottens formet som et sværd proces, armhulen og falciform ligament, og hjertesækken af ​​hjertet. Disse milepæle er vigtigt at øge succesen af ​​proceduren og for at sikre et højere udbytte af hjertets immunceller. Disse isolerede hjerte immunceller kan derefter bruges til karakterisering af funktionalitet, fænotype, modenhed, og co-kultur eksperimenter med andre hjerteceller at få en bedre forståelse af deres samspil.

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

Denne metode til at isolere funktionel immunceller fra hjertet giver et alternativ til de konventionelle metoder collagenase fordøjelse, der forårsager uønsket immun celle aktivering, hvilket resulterer i en nedsat reaktionsevne af disse celler. Vores metode til isolation giver funktionelle hjertets immunceller ved at undgå problemer i forbindelse med enzymatisk fordøjelse.

Isolering af Funktionel Cardiac immunceller

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

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