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. Isolement et culture de cellules endothéliales humaines primaires et Cellules souches mésenchymateuses <ol><li> cellules d&#39;isolement et de la culture des droits de l&#39;endothéliales de veine ombilicale (HUVEC): <ol><li> Mettez cordon ombilical sur un plateau avec des serviettes en papier et pulvériser avec 70% d&#39;éthanol. Placez dans une hotte de culture de tissu. Identifier la veine et Cathétériser aux deux extrémités. Placez une attache de câble autour de l&#39;extrémité canule pour le fixer....

<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>

Nous décrivons ici deux vitro &quot;vasculaires&quot; modèles pour étudier le recrutement de neutrophiles circulants par l&#39;endothélium enflammé. Un avantage majeur de ces modèles est la capacité d&#39;analyser chaque étape de la cascade d&#39;adhérence des leucocytes dans l&#39;ordre, comme cela se produirait in vivo. Nous avons déjà observé une augmentation dose-dépendante de l&#39;adhésion des neutrophiles à travers transmigration et stimulée par le TNFa CE 9,29.</su...

L&#39;inflammation est une réponse protectrice à une infection microbienne ou d&#39;une blessure de tissu qui nécessite une réglementation stricte de l&#39;entrée de leucocytes et la sortie de l&#39;inflammation des tissus pour permettre la résolution 1,2. Diaphonie entre les cellules endothéliales (CE) que les vaisseaux sanguins de la ligne, leucocytes circulants et les cellules stromales tissus-résident est essentiel de coordonner ce processus 3. Cependant, le recrutement incontrôlée de leucocytes et de leur dédouanement inefficaces sous-tendent le développement de maladies inflammatoires chroniques 4. Notre compréhension actuelle de recrutement des leucocytes dans la santé et la maladie est incomplète et modèles plus robustes sont nécessaires pour analyser ce processus. Les mécanismes de soutien au recrutement de leucocytes du sang à travers vasculaire CE veinules post-capillaires ont été bien décrits 1,2,5. Leucocytes circulants sont capturés par des récepteurs spécialisés (par exemple, VCAM-1, E-sélectine, la P-sélectine) quisont régulés à la hausse sur l&#39;endothélium enflammé. Ces interactions transitoires permettent d&#39;interagir avec les leucocytes surface lié chimiokines et médiateurs dérivés de lipides (soit stromales endotheliales ou d&#39;origine) qui activent les intégrines exprimées par les leucocytes 6- 11. Cela stabilise l&#39;adhérence et la migration disques à travers l&#39;endothélium et dans le tissu 12 15. Dans le tissu, recrutés leucocytes sont soumis à des agents stromales dérivées qui influencent leur motilité, la fonction et la survie 16,17. L&#39;évidence croissante suggère fortement que les signaux reçus à chaque étape des conditions de processus de recrutement des leucocytes pour la prochaine. Cependant, notre compréhension de recrutement des leucocytes reste incomplète et très peu est connu sur le mouvement des leucocytes composants de mise en forme dans le tissu. A Birmingham, nous avons développé plusieurs in vitro &quot;vasculaires&quot; modèles pour étudier le recrutement de leucocytes9,18,19 écouler. Nous comprenons maintenant que vasculaire acte CE régulateurs immédiats de recrutement des leucocytes répondre aux changements dans leur micro-environnement local. Plus précisément, les cellules stromales tissus-résident peuvent activement réguler la réponse inflammatoire, en partie en conversant avec vasculaire CE voisine d&#39;influencer leur rôle dans le recrutement 3. Nous avons précédemment montré que diverses cellules stromales modulent la capacité de CE pour soutenir l&#39;adhérence et la migration des leucocytes d&#39;une manière spécifique d&#39;un tissu, et que ces effets deviennent altérée dans les maladies chroniques 13,16,20,21. Ainsi, les cellules stromales établissent tissus &#39;adresse-codes »qui définissent le contexte de chaque réponse inflammatoire 22. Plus récemment, nous avons démontré que la moelle osseuse provenant MSC (BMMSC) puissamment réguler à la baisse la réponse des CE à des cytokines, conduisant à une réduction dans le recrutement de deux neutrophiles et de lymphocytes 23. Les mécanismes GOVrecrutement Erning élucidé in vitro ont largement utilisé les essais comportant un seul type de cellule (par exemple, CE) ou de protéines dans l&#39;isolement. Cependant, ces études ne prennent pas en considération les effets de l&#39;environnement tissulaire locale (ce est à dire, la présence de cellules stromales) sur le recrutement des leucocytes et leur migration ultérieure dans le tissu. Nous décrivons ici deux méthodes basées sur les flux dans lequel les cellules stromales, cellules souches mésenchymateuses spécifiquement (MSC), sont co-cultivées avec des CE 23. Ces modèles nous permettent d&#39;examiner l&#39;effet des cellules stromales sur les réponses endothéliales, en particulier leur capacité à soutenir le recrutement des leucocytes de l&#39;écoulement.

<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

Les cellules stromales réglementer le recrutement de leucocytes circulants cours de l&#39;inflammation par la diaphonie avec des cellules endothéliales voisins. Nous décrivons ici deux vitro &quot;vasculaires&quot; modèles pour étudier le recrutement de neutrophiles circulants de flux par les cellules endothéliales enflammées. Un avantage majeur de ces modèles est la capacité d&#39;analyser chaque étape de la cascade d&#39;adhérence des leucocytes dans l&#39;ordre, comme cela se produirait in vivo. Nous décrivons également comment les deux modèles peuvent être adaptés à étudier le rôle des cellules stromales, dans ce cas cellules souches mésenchymateuses (CSM), dans la régulation de recrutement des leucocytes. Cellules endotheliales primaires ont été cultivées seules ou conjointement avec MSC humain en contact direct sur microplaques Ibidi ou sur des côtés opposés d&#39;un filtre Transwell pendant 24 heures. Les cultures ont été stimulées avec le facteur de nécrose tumorale alpha (TNFa) pendant 4 heures et incorporés dans un essai d&#39;adhérence flow-based. Un bolus de neutrophiles a été perfusésur l&#39;endothélium pendant 4 min. La capture de neutrophiles et de leurs interactions avec l&#39;endothélium fluide a été visualisé par microscopie à contraste de phase. Dans les deux modèles, les cytokines stimulation augmente le recrutement de neutrophiles endothéliale se écoulant d&#39;une manière dépendante de la dose. L&#39;analyse du comportement des neutrophiles recrutés ont montré une diminution dose-dépendante de roulement et une augmentation dose-dépendante de la transmigration à travers l&#39;endothélium. En co-culture, MSC supprimé l&#39;adhésion des neutrophiles à l&#39;endothélium de TNFa-stimulé. Nos modèles d&#39;adhésion basés sur le débit imitent les phases initiales de recrutement des leucocytes de la circulation. En plus de leucocytes, ils peuvent être utilisés pour examiner le recrutement d&#39;autres types de cellules, comme les cellules tumorales circulantes MSC ou administrés thérapeutiquement. Notre modèle de co-culture multi-couches ont montré que les MSC communique avec endothélium de modifier leur réponse à des cytokines pro-inflammatoires, Altering le recrutement des neutrophiles. D&#39;autres recherches utilisant ces modèles est nécessaire pour bien comprendre comment les cellules stromales de différents tissus et les conditions (troubles inflammatoires ou de cancer) influencer le recrutement des leucocytes pendant l&#39;inflammation.

Initialement, nous avons analysé l&#39;effet de stimuler la CE avec du TNFa sur le recrutement de neutrophiles de flux en utilisant le modèle de microplaque Ibidi (article 7-9). En l&#39;absence de TNFa, peu ou pas de neutrophiles ont adhéré à la monocouche endotheliale (figure 2A). Ceci a été prévu, en tant que non traité / CE repos ne expriment pas les molécules d&#39;adhérence nécessaires (les sélectines) ou à l&#39;appui de liaison de chimiokines 25,26. En r...

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

La capacité de l&#39;endothélium enflammé de recruter leucocytes du débit est régulé par les cellules stromales mésenchymateuses. Nous décrivons deux modèles in vitro incorporant cellules humaines primaires qui peuvent être utilisés pour évaluer le recrutement des neutrophiles de flux et d&#39;examiner le rôle que les cellules stromales mésenchymateuses jouent dans la régulation de ce processus.

Analyser les effets des cellules stromales sur le recrutement des leucocytes de débit

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

Isolement et analyse de Brain-séquestrés de leucocytes<em&gt; Plasmodium berghei</em&gt; ANKA-souris infectées

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

Recherche

Immunologie et infection

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

Étude des effets des probiotiques sur la colonisation pneumococcique l&#39;aide d&#39;un<em&gt; In Vitro</em&gt; Respect de dosage

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

Isolement des leucocytes à partir de l&#39;interface humaine materno-fœtale

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

Isolement de leucocytes des tissus murins à l&#39;interface materno-foetale

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

L&#39;utilisation de la cytométrie de flux pour évaluer l&#39;état de la chromatine dans les cellules T

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

Isolement et analyse par cytométrie en flux des cellules immunitaires du cerveau ischémique souris

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

Fluorescence-tri cellulaire activé pour la purification de cellules dendritiques plasmacytoïdes de la moelle osseuse de souris

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

Cytométrie en flux Analyse de la particule liée Bet v 1 Allergène en 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

Visualisation des effets des expectorations sur le développement du biofilm Utilisation d&#39;un modèle Chambered lamelle couvre-objet

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

Analyses d’enquêter sur le recrutement des leucocytes sur les cellules vasculaires et plaquettes adhérentes à flux laminaire

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