Name: analyzing the effects of stromal cells on the recruitment of leukocytes from flow
Uploaded: 2015-01-07T15:00:00
Description: Watch this Scientific Journal Video about Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow at JoVE.com

Umbilical cords were collected with the assistance of the Birmingham Women's Health Care NHS Trust. HMM was supported by an Arthritis Research UK Career Development Fellowship (19899) and Systems Science for Health, University of Birmingham (5212).

1. Isolering og Culture of Primary humane endotelceller og stamceller <ol><li> Isolasjon og kultur av menneskelige navleveneendotel celler (HUVEC): <ol><li> Sett navlestreng på et brett med tørkepapir og spray med 70% etanol. Plassere i en vev kultur hette. Identifisere venen og cannulate i begge ender. Plasser en strips rundt cannulated slutt å sikre det. </li><li> Vask med venøst ​​blod med PBS ved anvendelse av en sprøyte. Fyll sprøyten med luft og passerer gjennom venen å ta bort den gjenværende PBS. <...

<ol>
	<li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+on+endothelium+for+lymphocyte+recirculation+and+leukocyte+emigration.">Traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration.</a> Ann. Rev. Physiol. 57, 827-872 (1995).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nature reviews. Immunology. 7 (9), 678-689 (2007).</li><li>McGettrick, H. M., Butler, L. M., Buckley, C. D., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+stroma+as+a+regulator+of+leukocyte+recruitment+in+inflammation.">Tissue stroma as a regulator of leukocyte recruitment in inflammation.</a> Journal of leukocyte biology. 91 (3), 385-400 (2012).</li><li>Serhan, C. N., Savill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolution+of+inflammation:+the+beginning+programs+the+end.">Resolution of inflammation: the beginning programs the end.</a> Nature immunology. 6 (12), 1191-1197 (2005).</li><li>Schmidt, S., Moser, M., Sperandio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+leukocyte+recruitment+and+its+deficiencies.">The molecular basis of leukocyte recruitment and its deficiencies.</a> Molecular immunology. 55, 49-58 (2013).</li><li>Luu, N. T., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Ability+of+Exogenous+Chemotactic+Agents+to+Disrupt+Transendothelial+Migration+of+Flowing+Neutrophils.">Differential Ability of Exogenous Chemotactic Agents to Disrupt Transendothelial Migration of Flowing Neutrophils.</a> The Journal of Immunology. 164 (11), 5961-5969 (2000).</li><li>Smith, C. W., Rothlein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+an+endothelial+determinant+for+CD+18-dependent+human+neutrophil+adherence+and+transendothelial+migration.">Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration.</a> The Journal of clinical investigation. 82 (5), 1745-1756 (1988).</li><li>Luscinskas, F. W., Brock, A. F., Arnaout, M. A., Gimbrone, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-leukocyte+adhesion+molecule-1-dependent+and+leukocyte+(CD11/CD18)-dependent+mechanisms+contribute+to+polymorphonuclear+leukocyte+adhesion+to+cytokine-activated+human+vascular+endothelium.">Endothelial-leukocyte adhesion molecule-1-dependent and leukocyte (CD11/CD18)-dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine-activated human vascular endothelium.</a> J. Immunol. 142 (7), (1989).</li><li>Bahra, P., Rainger, G. E., Wautier, J. L., Nguyet-Thin, L., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Each+step+during+transendothelial+migration+of+flowing+neutrophils+is+regulated+by+the+stimulatory+concentration+of+tumour+necrosis+factor-alpha.">Each step during transendothelial migration of flowing neutrophils is regulated by the stimulatory concentration of tumour necrosis factor-alpha.</a> Cell adhesion and communication. 6 (6), 491-501 (1998).</li><li>Piali, L., Weber, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+receptor+CXCR3+mediates+rapid+and+shear-resistant+adhesion-induction+of+effector+T+lymphocytes+by+the+chemokines+IP10+and+Mig.">The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig.</a> European journal of immunology. 28 (3), 961-972 (1998).</li><li>McGettrick, H. M., Smith, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+from+different+sites+may+promote+or+inhibit+recruitment+of+flowing+lymphocytes+by+endothelial+cells.">Fibroblasts from different sites may promote or inhibit recruitment of flowing lymphocytes by endothelial cells.</a> European journal of immunology. 39 (1), 113-125 (2009).</li><li>McGettrick, H. M., Hunter, K., Moss, P. a., Buckley, C. D., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observations+of+the+kinetics+of+migrating+T+cells+suggest+active+retention+by+endothelial+cells+with+continual+bidirectional+migration.">Direct observations of the kinetics of migrating T cells suggest active retention by endothelial cells with continual bidirectional migration.</a> Journal of leukocyte biology. 85 (1), 98-107 (2009).</li><li>McGettrick, H. M., Buckley, C. D., Filer, A., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cells+differentially+regulate+neutrophil+and+lymphocyte+recruitment+through+the+endothelium.">Stromal cells differentially regulate neutrophil and lymphocyte recruitment through the endothelium.</a> Immunology. 131 (3), 357-370 (2010).</li><li>Tull, S. P., Yates, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Omega-3+Fatty+acids+and+inflammation:+novel+interactions+reveal+a+new+step+in+neutrophil+recruitment.">Omega-3 Fatty acids and inflammation: novel interactions reveal a new step in neutrophil recruitment.</a> PLoS biology. 7 (8), e1000177 (2009).</li><li>Ahmed, S. R., McGettrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+D2+regulates+CD4++memory+T+cell+trafficking+across+blood+vascular+endothelium+and+primes+these+cells+for+clearance+across+lymphatic+endothelium.">Prostaglandin D2 regulates CD4+ memory T cell trafficking across blood vascular endothelium and primes these cells for clearance across lymphatic endothelium.</a> Journal of immunology. 187 (3), 1432-1439 (2011).</li><li>Bradfield, P. F., Amft, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheumatoid+fibroblast-like+synoviocytes+overexpress+the+chemokine+stromal+cell-derived+factor+1+(CXCL12),+which+supports+distinct+patterns+and+rates+of+CD4++and+CD8++T+cell+migration+within+synovial+tissue.">Rheumatoid fibroblast-like synoviocytes overexpress the chemokine stromal cell-derived factor 1 (CXCL12), which supports distinct patterns and rates of CD4+ and CD8+ T cell migration within synovial tissue.</a> Arthritis and rheumatism. 48 (9), 2472-2482 (2003).</li><li>Filer, A., Parsonage, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+survival+of+leukocyte+subsets+mediated+by+synovial,+bone+marrow,+and+skin+fibroblasts:+site-specific+versus+activation-dependent+survival+of+T+cells+and+neutrophils.">Differential survival of leukocyte subsets mediated by synovial, bone marrow, and skin fibroblasts: site-specific versus activation-dependent survival of T cells and neutrophils.</a> Arthritis and rheumatism. 54 (7), 2096-2108 (2006).</li><li>Lally, F., Smith, E., et al. <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+mechanism+of+neutrophil+recruitment+in+a+coculture+model+of+the+rheumatoid+synovium.">A novel mechanism of neutrophil recruitment in a coculture model of the rheumatoid synovium.</a> Arthritis and rheumatism. 52 (11), 3460-3490 (2005).</li><li>Chakravorty, S. J., McGettrick, H. M., Butler, L. M., Buckley, C. D., Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro.+model+for+analysing+neutrophil+migration+into+and+away+from+the+sub-endothelial+space:+Roles+of+flow+and+CD31.">An in vitro. model for analysing neutrophil migration into and away from the sub-endothelial space: Roles of flow and CD31.</a> Biorheology. 43 (1), 71-82 (2006).</li><li>Rainger, G. E., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Pathology+of+Atherosclerosis+Smooth+Muscle+Cells+Prime+Cocultured+Endothelial+Cells+for+Enhanced+Leukocyte+Adhesion.">Cellular Pathology of Atherosclerosis Smooth Muscle Cells Prime Cocultured Endothelial Cells for Enhanced Leukocyte Adhesion.</a> Circulation Research. 88 (6), 615-622 (2001).</li><li>Kuravi, S. J., McGettrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Podocytes+regulate+neutrophil+recruitment+by+glomerular+endothelial+cells+via+IL-6-mediated+crosstalk.">Podocytes regulate neutrophil recruitment by glomerular endothelial cells via IL-6-mediated crosstalk.</a> Journal of immunology. 193 (1), 234-243 (2004).</li><li>Parsonage, G., Filer, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stromal+address+code+defined+by+fibroblasts.">A stromal address code defined by fibroblasts.</a> Trends in immunology. 26 (3), 150-156 (2005).</li><li>Luu, N. T., McGettrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+mesenchymal+stem+cells+and+endothelial+cells+leads+to+downregulation+of+cytokine-induced+leukocyte+recruitment.">Crosstalk between mesenchymal stem cells and endothelial cells leads to downregulation of cytokine-induced leukocyte recruitment.</a> Stem cells. 31 (12), 2690-2702 (2013).</li><li>Bevilacqua, M. P., Nelson, R. M., Mannori, G., Cecconi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-leukocyte+adhesion+molecules+in+human+disease.">Endothelial-leukocyte adhesion molecules in human disease.</a> Annual review of medicine. 45, 361-378 (1994).</li><li>Stanness, K. A., Beatty, P. G., Ochs, H. D., Harlan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+endothelial+cell+surface+factor(s)+induced+in+vitro.">An endothelial cell surface factor(s) induced in vitro.</a> 136 (12), 4548-4553 (1986).</li><li>Burton, V. J., Butler, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delay+of+migrating+leukocytes+by+the+basement+membrane+deposited+by+endothelial+cells+in+long-term+culture.">Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture.</a> Experimental cell research. 317 (3), 276-292 (2011).</li><li>Luu, N. T., Rainger, G. E., Buckley, C. D., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD31+Regulates+Direction+and+Rate+of+Neutrophil+Migration+over+and+under+Endothelial+Cells.">CD31 Regulates Direction and Rate of Neutrophil Migration over and under Endothelial Cells.</a> Journal of Vascular Research. 40 (5), 467-479 (2003).</li><li>McGettrick, H. M., Buckley, C. D., Ed Rainger, G., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+stromal+cells+on+lymphocyte+adhesion+and+migration+on+endothelial+cells.">Influence of stromal cells on lymphocyte adhesion and migration on endothelial cells.</a> Methods in molecular biology. 616, 49-68 (2010).</li><li>Butler, L. M., McGettrick, H. M., Nash, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Static+and+dynamic+assays+of+cell+adhesion+relevant+to+the+vasculature.">Static and dynamic assays of cell adhesion relevant to the vasculature.</a> Methods in molecular biology. 467, 211-228 (2009).</li><li>Jeffery, H. C., Buckley, C. D., Moss, P., Rainger, G. E., Nash, G. B., McGettrick, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+effects+of+stromal+cells+on+the+migration+of+lymphocytes+into+and+through+inflamed+tissue+using+3-D+culture+models.">Analysis of the effects of stromal cells on the migration of lymphocytes into and through inflamed tissue using 3-D culture models.</a> Journal of immunological methods. 400-401, 45-57 (2013).</li></ol>

Her beskriver vi to in vitro &quot;vaskulære&quot; modeller for å studere rekruttering av sirkulerende nøytrofile ved betent endotelet. En stor fordel ved disse modellene er evnen til å analysere hvert trinn i leukocyttadhesjon kaskade i rekkefølge, som ville forekomme in vivo. Vi har tidligere observert en doseavhengig økning i nøytrofil adhesjon til og transmigrasjon gjennom TNFa-stimulerte EC 9,29. Vi beskriver også hvordan begge modellene kan tilpasses for å studere effektene av...

Betennelse er en beskyttende respons på mikrobiell infeksjon eller vevsskade som krever tett regulering av leukocytt-inngang og utgang fra det betente vevet for å tillate oppløsning 1,2. Krysstale mellom endotelceller (EC) at linjen blodkar, utegående leukocytter og vev-bosatt stromalceller er viktig for å koordinere denne prosessen tre. Men ukontrollert rekruttering av leukocytter og deres ineffektive klaring ligger til grunn for utvikling av kroniske betennelsessykdommer 4. Vår nåværende forståelse av leukocytt rekruttering i helse og sykdom er ufullstendig og mer robuste modeller for å analysere denne prosessen. Mekanismene som støtter rekruttering av leukocytter fra blod gjennom vaskulær EC i post-kapillære venuler har blitt godt beskrevet 1,2,5. Sirkulerende leukocytter fanges opp av spesialiserte reseptorer (f.eks VCAM-1, E-selectin, P-selektinantistoffer) somer oppregulert på betent endotelet. Disse forbigående interaksjoner tillate leukocytter å samhandle med overflate bundet kjemokiner og lipid-avledet formidlere (enten endotelceller eller stromal opprinnelse) som aktiverer inte uttrykt av leukocytter 6- 11. Dette i sin tur stabiliserer adhesjon og migrasjon stasjoner over endotelet og inn i vevet 12- 15. Innenfor vev, leukocytter rekrutteres utsettes for stromal-avledet midler som påvirker deres motilitet, funksjon og overlevelse 16,17. Økende bevis tyder på at signalene som mottas på hvert trinn i rekrutteringsprosessen vilkår leukocytter for den neste. Men vår forståelse av leukocytt rekruttering er fortsatt ufullstendig og svært lite er kjent om komponentene forme leukocytt bevegelse innenfor vev. I Birmingham har vi utviklet flere in vitro &quot;vaskulære&quot; modeller for å studere rekruttering av leukocytter fraflyte 9,18,19. Nå forstår vi at vaskulær EC fungere som umiddelbare regulatorer av leukocytt rekruttering ta hensyn til endringer i deres lokale mikromiljøet. Nærmere bestemt, kan vev-resident stromalceller aktivt regulere betennelsesreaksjon, blant annet ved samtale med nabo vaskulær EC til å påvirke sin rolle i rekruttering tre. Vi har tidligere vist at forskjellige stromale celler modulerer evnen av EC å støtte adhesjon og migrasjon av leukocytter i en vev-spesifikk måte, og at disse virkninger blir endret i kroniske sykdommer 13,16,20,21. Dermed stromalceller etablere vev &#39;adresse-koder&#39; som definerer konteksten av hver betennelsesreaksjon 22. Mer nylig, har vi vist at benmargs avledet MSC (BMMSC) potent nedregulerer responsen av EC til cytokiner, fører til en reduksjon i rekruttering av både neutrofiler og lymfocytter 23. Mekanismene governing rekruttering belyst in vitro har i stor grad brukes analyser innlemme et enkelt celletype (f.eks EF) eller protein i isolasjon. Men disse studier ikke ta hensyn til virkningene av lokale vev miljø (dvs. tilstedeværelse av stromale celler) på rekrutteringen av leukocytter og deres påfølgende migrasjon inn i vevet. Her beskriver vi to strømningsbaserte metoder hvor stromalceller, spesielt mesenchymale stamceller (MSC), er co-kultivert med EC 23. Slike modeller tillate oss å undersøke effekten av stromal celle på endotelceller svar, spesielt deres evne til å støtte leukocytt rekruttering fra flyt.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Collagenase Type Ia</td>
			<td >Sigma</td>
			<td>C2674</td>
			<td>Dilute in 10ml PBS to get a final concentration of 10mg/ml. Store at -20&#176;C in 1ml aliquots.</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s PBS</td>
			<td >Sigma</td>
			<td >D8662</td>
			<td>With calcium and magnesium chloride. Keep sterile and store at room temperature.</td>
		</tr>
		<tr >
			<td >1X Medium M199</td>
			<td >Gibco</td>
			<td >31150-022</td>
			<td>Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr >
			<td >Gentamicin sulphate</td>
			<td >Sigma</td>
			<td >G1397</td>
			<td>Store at 4&#176;C. Add to M199 500ml bottle.</td>
		</tr>
		<tr >
			<td >Human epidermal growth factor</td>
			<td >Sigma</td>
			<td >E9644</td>
			<td>Store at -20&#176;C in 10&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >Fetal calf serum (FCS)</td>
			<td >Sigma</td>
			<td >F9665</td>
			<td >FCS must be batch tested to ensure the growth and viability of isolated EC. Heat inactivate at 56&#176;C. Store in 10ml aliquots at -20&#176;C.</td>
		</tr>
		<tr >
			<td >Amphotericin B</td>
			<td >Gibco</td>
			<td >15290-026</td>
			<td >Potent and becomes toxic within a week so fresh complete HUVEC medium must be made up every week. Store at -20&#176;C in 1ml aliquots.</td>
		</tr>
		<tr >
			<td >Hydrocortisone</td>
			<td >Sigma</td>
			<td >H0135</td>
			<td>Stock is in ethanol. Store at -20&#176;C in 10&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >Collagenase Type II</td>
			<td >Sigma</td>
			<td >C6885</td>
			<td>Dilute stock in PBS to a final concentration of 100mg/ml. Store at -20&#176;C in 100&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >Hyaluronidase</td>
			<td >Sigma</td>
			<td >H3631</td>
			<td>Dilute stock in PBS to a final concentration of 20,000U/ml. Store at -20&#176;C in 100&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >100&#181;m cell strainer for 50ml centifuge tube</td>
			<td >Scientific Lab Supplies (SLS)</td>
			<td >352360</td>
			<td >Other commercially available cell strainers (e.g. Greiner bio-one) can also be used.</td>
		</tr>
		<tr >
			<td >DMEM low glucose</td>
			<td >Biosera</td>
			<td >LM-D1102/500</td>
			<td>Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr >
			<td >Penicillin/Streptomycin mix</td>
			<td >Sigma</td>
			<td >P4333</td>
			<td>Store at -20&#176;C in 1ml aliquots.</td>
		</tr>
		<tr >
			<td >25cm2 tissue culture flask</td>
			<td >SLS</td>
			<td >353109</td>
			<td></td>
		</tr>
		<tr >
			<td >75cm2 tissue culture flask</td>
			<td >SLS</td>
			<td >353136</td>
			<td></td>
		</tr>
		<tr >
			<td >Bone marrow mesenchymal stem cells vial</td>
			<td >Lonza</td>
			<td >PT-2501</td>
			<td >Store in liquid nitrogen upon arrival. Cells are at passage 2 upon arrival but are designated passage 0. Exapand to passage 3 and store in liquid nitrogen for later use.</td>
		</tr>
		<tr >
			<td >Mesenchymal stem cell growth medium (MSCGM)</td>
			<td >Lonza</td>
			<td >PT-3001</td>
			<td>Warm in 37 &#176;C water bath before use. For Cell Tracker Green staining use medium without FCS.</td>
		</tr>
		<tr >
			<td >EDTA (0.02%) solution</td>
			<td >Sigma</td>
			<td >E8008</td>
			<td>Store at 4&#176;C. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr >
			<td >Trypsin solution</td>
			<td >Sigma</td>
			<td >T4424</td>
			<td>Store at -20&#176;C in 2ml aliquots. Thaw at room temperature and use immediately.</td>
		</tr>
		<tr >
			<td >Cryovials</td>
			<td >Greiner bio-one</td>
			<td>2019-02</td>
			<td>Keep on ice before adding before adding cell suspension.</td>
		</tr>
		<tr >
			<td >Mr. Frosty Freezing Container</td>
			<td >Nalgene</td>
			<td >5100-0001</td>
			<td >Store at room temperature. When adding cryovials with cells store at -80&#176;C for 24h before transfrring cells to liquid nitrogen.</td>
		</tr>
		<tr >
			<td >Ibidi u-Slide VI (0.4), T/C treated, sterile</td>
			<td >Ibidi</td>
			<td >IB-80606</td>
			<td >Alternative models include glass capillaries, Cellix Biochips (www.cellixltd.com), BioFlux Plates (www.fluxionbio.com/bioflux/) and GlycoTech parallel plate flow chambers (http://www.glycotech.com/apparatus/parallel.html).</td>
		</tr>
		<tr >
			<td >Cell tracker green dye</td>
			<td >Life technologies</td>
			<td >C2925</td>
			<td>Store in 5&#181;l aliquots at -20&#176;C. Dilute in 5ml prewarmed (at 37&#176;C) MSCGM.</td>
		</tr>
		<tr >
			<td >Cell counting chambers</td>
			<td >Nexcelom</td>
			<td >SD-100</td>
			<td>Alternatively a haemocytometer can be used.</td>
		</tr>
		<tr >
			<td >Cellometer auto T4 cell counter</td>
			<td >Nexcelom</td>
			<td >Auto T4-203-0238</td>
			<td></td>
		</tr>
		<tr >
			<td >Tumor necrosis factor &#945; (TNF&#945;)</td>
			<td >R&#38;D Systems</td>
			<td >210-TA-100</td>
			<td>Dilute stock in PBS to a final concentration of 100,000U/ml. Store at -80&#176;C in 10&#181;l aliquots.</td>
		</tr>
		<tr >
			<td >6-well, 0.4&#181;m PET Transwell filters</td>
			<td >SLS</td>
			<td >353090</td>
			<td></td>
		</tr>
		<tr >
			<td >K2-EDTA in 10ml tubes</td>
			<td >Sarstedt</td>
			<td ></td>
			<td>Store at room temperature.</td>
		</tr>
		<tr >
			<td >Histopaque 1119</td>
			<td >Sigma</td>
			<td >11191</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr >
			<td >Histopaque 1077</td>
			<td >Sigma</td>
			<td >10771</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr >
			<td >10ml round bottomed tube</td>
			<td >Appleton Woods</td>
			<td >SC211 142 AS</td>
			<td></td>
		</tr>
		<tr >
			<td >7.5% BSA Fraction V solution</td>
			<td >Life technologies</td>
			<td >15260-037</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr >
			<td >20ml Plastipak syringes</td>
			<td >BD falcon</td>
			<td >300613</td>
			<td></td>
		</tr>
		<tr >
			<td >5ml Plastipak syringes</td>
			<td >BD falcon</td>
			<td >302187</td>
			<td></td>
		</tr>
		<tr >
			<td >2ml Plastipak syringes</td>
			<td >BD falcon</td>
			<td >300185</td>
			<td></td>
		</tr>
		<tr >
			<td >3M hypo-allergenic surgical tape 9m x 2.5cm</td>
			<td >Micropore</td>
			<td >1530-1</td>
			<td>Use to secure the syringe tap onto the wall of the perspex chamber.</td>
		</tr>
		<tr >
			<td >Silicon rubber tubing, internal diameter/external diameter (ID/OD) of 1/3mm (thin tubing)</td>
			<td >Fisher Scientific</td>
			<td >FB68854</td>
			<td >Cut silicon tubing to the appropriate size. All tubing leading directly to the electronic microvalve must be thin.</td>
		</tr>
		<tr >
			<td >Silicon rubber tubing ID/OD of 2/4mm (thick tubing)</td>
			<td >Fisher Scientific</td>
			<td >FB68855</td>
			<td></td>
		</tr>
		<tr >
			<td >Portex Blue Line Manometer tubing</td>
			<td>Smiths</td>
			<td >200/495/200</td>
			<td>Tubing leading to the syringe pump.</td>
		</tr>
		<tr >
			<td >3-way stopcock</td>
			<td >BOC Ohmeda AB</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Glass 50ml syringe for pump</td>
			<td >Popper Micromate</td>
			<td >550962</td>
			<td>Must be primed prior to use by removing any air bubbles.</td>
		</tr>
		<tr >
			<td >Glass coverslip</td>
			<td >Raymond A Lamb</td>
			<td ></td>
			<td>26x76mm coverslips made to order. Lot number 2440980.</td>
		</tr>
		<tr >
			<td >Parafilm gasket</td>
			<td >American National Can Company</td>
			<td ></td>
			<td >Cut a 26x76mm piece of parafilm using an aluminium template and cut a 20x4mm slot into it using a scalpel 10a. Gasket thickness is approximately 133&#181;m.</td>
		</tr>
		<tr >
			<td >Two perspex parallel plates</td>
			<td >Wolfson Applied Technology Laboratory</td>
			<td ></td>
			<td >Specially designed chamber consisting of parallel plates held together by 8 screws. The lower plate has a viewing slot cut out in the middle and a shallow recess milled to allow space for the coverslip, filter and gasket. The upper perspex plate has an inlet and outlet hole positioned over the flow channel.</td>
		</tr>
		<tr >
			<td >Electronic 3-way microvalve with min. dead space</td>
			<td >Lee Products Ltd.</td>
			<td >LFYA1226032H</td>
			<td>Electronically connected to a 12 volt DC power supply.</td>
		</tr>
		<tr >
			<td >Syringe pump for infusion/withdrawal (PHD2000)</td>
			<td >Harvard Apparatus</td>
			<td >70-2001</td>
			<td>Set the diameter to 29mm and refill (flow) rate.</td>
		</tr>
		<tr >
			<td >L-shaped connector</td>
			<td >Labhut</td>
			<td >LE876</td>
			<td>To attach to the inlet and outlet ports onto the Ibidi microslide channel.</td>
		</tr>
		<tr >
			<td >Video camera</td>
			<td >Qimaging</td>
			<td >01-QIC-F-M-12-C</td>
			<td>Connected to a computer which enables digitall videos to be recorded.</td>
		</tr>
		<tr >
			<td >Image-Pro Plus 7.0</td>
			<td >Media Cybernetics</td>
			<td >41N70000-61592</td>
			<td >For data analysis. Manually tag cells displaying the different behaviors. Track cells for analysis of rolling and migration velocities.</td>
		</tr>
		<tr >
			<td ></td>
			<td ></td>
			<td ></td>
			<td>Refer to product datasheets for details on hazards of using the reagents described here.</td>
		</tr>
	</tbody>
</table>

analyzing the effects of stromal cells on the recruitment of leukocytes from flow

Stromalceller regulere rekrutteringen av sirkulerende leukocytter under betennelse gjennom krysstale med nabo endotelceller. Her beskriver vi to in vitro &quot;vaskulære&quot; modeller for å studere rekruttering av sirkulerende nøytrofile granulocytter fra flyten av betente endotelceller. En stor fordel ved disse modellene er evnen til å analysere hvert trinn i leukocyttadhesjon kaskade i rekkefølge, som ville forekomme in vivo. Vi beskriver også hvordan begge modellene kan tilpasses for å studere rollen til stromale celler, i dette tilfellet mesenchymale stamceller (MSC), i regulering av leukocytt-rekruttering. Primære endotelceller ble dyrket alene eller sammen med human MSC i direkte kontakt på Ibidi microslides eller på motsatte sider av en Transwell filter i 24 timer. Kulturene ble stimulert med tumornekrosefaktor alfa (TNFa) i 4 timer og innlemmet i en strømningsbasert adhesjonsanalysen. En bolus av nøytrofile ble perfusertover endotelet i 4 min. Fangst av rennende nøytrofile og deres samspill med endotelet ble visualisert ved fasekontrastmikroskopi. I begge modellene, økt cytokin-stimulering endothelial rekruttering av strømmer nøytrofile på en doseavhengig måte. Analyse av oppførselen til rekrutteres neutrofiler viste en doseavhengig reduksjon i rullende og en doseavhengig økning i transmigrasjon gjennom endotelet. I co-kultur, MSC trykt nøytrofiladhesjon til TNFa-stimulert endotelet. Våre flytbasert-vedheft modeller ligne de innledende fasene av leukocytt rekruttering fra sirkulasjonen. I tillegg til leukocytter, kan de anvendes til å undersøke rekrutteringen av andre celletyper, slik som terapeutisk administrerte MSC eller sirkulerende tumorceller. Våre flerlags co-kultur modeller har vist at MSC kommunisere med endotelet til å endre sin respons på proinflammatoriske cytokiner, altering rekruttering av nøytrofile. Videre forskning ved hjelp av slike modeller er nødvendig for å fullt ut forstå hvordan stromale celler fra ulike vev og betingelsene (inflammatoriske lidelser eller kreft) påvirker rekruttering av leukocytter ved betennelse.

I utgangspunktet analysert vi effekten av stimulerende EC med TNFa på rekruttering av nøytrofile fra flyt bruker Ibidi microslide modell (§ 7 - 9). I fravær av TNFa, liten eller ingen neutrofiler klebet til endotel-monolag (figur 2A). Dette var ventet, som ubehandlet / hviler EF uttrykker ikke de nødvendige adhesjonsmolekyler (selectins) eller chemokiner å støtte bindende 25,26. I motsetning til dette cytokin-stimulering betydelig økt nøytrofil adhesjon til endotel p...

Watch this Scientific Journal Video about Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow at JoVE.com

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Muligheten av betent endotel å rekruttere leukocytter fra strømning reguleres av mesenchymale stromale celler. Vi beskriver to in vitro-modeller som har primære humane celler som kan brukes til å vurdere neutrofil rekruttering fra strømningen og undersøke rollen som mesenchymale stromale celler spiller i å regulere denne prosessen.

Analysere effekten av stromalceller på rekruttering av leukocytter fra Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Research

JoVE Journal

Immunology and Infection

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

Isolering og analyse av Brain-sequestered Leukocytter fra<em&gt; Plasmodium berghei</em&gt; ANKA-infiserte mus

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Gransker effekten av Probiotika på Pneumokokk Colonization Bruke en<em&gt; In Vitro</em&gt; Overholdelse analysen

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Isolering av leukocytter fra Human Maternal-foster Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Isolering av leukocyttene fra Murine Vev på Maternal Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

Bruk av flowcytometrisystemer å vurdere tilstanden til Chromatin i T-celler

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Isolasjon og flowcytometrisk analyse av immunceller fra iskemisk Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescens-aktivert cellesortering for Rensing av Plasmacytoid dendrittiske celler fra mus Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flowcytometrisk analyse av partikkelbundet Bet v 1 Allergen i PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Visualisere effekten av Sputum på Biofilm Development Ved hjelp av en Chambered dekkglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

Laminær strømning-baserte analyser undersøke leukocytter rekruttering på kulturperler vaskulære celler og tilhenger blodplater

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...