Name: generation of human cd40-activated b cells
Uploaded: 2009-10-16T23:30:00
Description: Watch this Scientific Journal Video about Generation of Human CD40-activated B cells Video at JoVE.com

<p class="jove_content"> मानव CD40 सक्रिय PBMC से बी कोशिकाओं की पीढ़ी के लिए प्रोटोकॉल को दो भागों में बांटा गया है: भाग NIH/3T3 कोशिकाओं, जो थाली बाध्य फीडर कोशिकाओं के रूप में इस्तेमाल किया जाएगा व्यक्त CD40 ligand की तैयारी को दर्शाता है. पार...

<ol>
	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> <em style='font-style: italic'>J Clin Invest</em>. <b>100</b>, 2757-2765 (1997).</li><li>Schultze, J. L., Grabbe, S., vonBergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> <em style='font-style: italic'>Trends Immunol</em>. <b>25</b>, 659-664 (2004).</li><li>Bergwelt-Baildon, M. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> <em style='font-style: italic'>Blood</em>. <b>107</b>, 2786-2789 (2006).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> <em style='font-style: italic'>PLoS ONE</em>. <b>3</b>, 1464-14 (2008).</li><li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand+triggered+interleukin-6+secretion+in+multiple+myeloma.">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> <em style='font-style: italic'>Blood</em>. <b>85</b>, 1903-1912 (1995).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> <em style='font-style: italic'>Cancer Res</em>. <b>63</b>, 2836-2843 (2003).</li><li>von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> <em style='font-style: italic'>Blood</em>. <b>99</b>, 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> <em style='font-style: italic'>Clin Exp Immunol</em>. <b>155</b>, 249-256 (2009).</li><li>Mason, 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=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> <em style='font-style: italic'>Gene Ther</em>. <b>15</b>, 955-965 (2008).</li></ol>

<p class="jove_title"> मीडिया के ए तैयार: </p><table border="1"cellspacing="0"><tbody><tr><td colspan="2"> <strong>फीडर सेल जंगली प्रकार मध्यम</strong> </td><td colspan="2"> <strong>फीडर सेल चयन मध्यम</strong> </td></tr><tr><td> <strong>अभिकर्मक</strong> </td><td> <strong>एकाग्रता</strong> </td><td> <strong>अभिकर्मक</strong> </td><td> <strong>एकाग्रता</strong> </td></tr><tr><td> / DMEM - हाम F12 </td><td></td><td> / DMEM - हाम F12 </td><td></td></tr><tr><td> एल-Glutamine </td><td> 365 μg / एमएल </td><td> एल-Glutamine </td><td> 365 μg / एमएल </td></tr><tr><td> FBS </td><td> 10% </td><td> FBS </td><td> 10% </td></tr><tr><td> HEPES </td><td> 10 मिमी </td><td> HEPES </td><td> 10 मिमी </td></tr><tr><td> Gentamycin </td><td> 15 μg / एमएल </td><td> Gentamycin </td><td> 15 μg / एमएल </td></tr><tr><td></td><td></td><td> G-418 </td><td> 0.7 मिलीग्राम / एमएल </td></tr></table><table cellspacing="0" border="1"><tbody><tr><td colspan="2"> <strong>CD40 - बी धोने मध्यम</strong> </td><td colspan="2"> <strong>CD40 बी संस्कृति के माध्यम</strong> </td></tr><tr><td> <strong>अभिकर्मक</strong> </td><td> <strong>एकाग्रता</strong> </td><td> <strong>अभिकर्मक</strong> </td><td> <strong>एकाग्रता</strong> </td></tr><tr></tr></tbody><tbody><tr><td> IMDM </td><td><br /></td><td> IMDM </td><td><br /></td></tr></tbody><tbody><tr><td> एल-Glutamine </td><td> 584 μg / एमएल </td><td> एल-Glutamine </td><td> 584 μg / एमएल </td></tr></tbody><tbody><tr><td> HEPES </td><td> 25 मिमी </td><td> HEPES </td><td> 25 मिमी </td></tr></tbody><tbody><tr><td> Gentamycin </td><td> 15 μg / एमएल </td><td> Gentamycin </td><td> 15 μg / एमएल </td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> आरएच transferrin </td><td> 50 μg / एमएल </td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> आरएच इंसुलिन </td><td> 5 μg / एमएल </td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> अटल बिहारी Humanserum </td><td> 10% </td></tr></tbody></table><p class="jove_title"> बी विविध अभिकर्मकों: </p><table cellspacing="0" border="1"><tbody><tr><td> <strong>अभिकर्मक</strong> </td><td> <strong>कंपनी</strong> </td><td> <strong>कोई आदेश</strong> </td></tr><tr></tr><tr><td> DMEM / हाम है F12 </td><td> Paa </td><td> कैट नहीं: E15 813 </td></tr><tr><td> IMDM </td><td> Invitrogen Gibco ® </td><td> 21980-032 रेफरी </td></tr><tr><td> Dulbecco पीबीएस (10x) </td><td> Paa </td><td> नहीं H15-011 बिल्ली </td></tr><tr><td> अटल बिहारी - मानव सीरम </td><td> Invitrogen </td><td> 34005100 कोई बिल्ली </td></tr><tr><td> मानव Holo - transferrin </td><td> सिग्मा </td><td> T0665 कोई बिल्ली </td></tr><tr><td> इंसुलिन मानव </td><td> सिग्मा </td><td> I2643 कोई बिल्ली </td></tr><tr><td> Gencin ® </td><td> डेल्टा चुनें </td><td> 7395800 कोई कला </td></tr><tr><td> पुनः संयोजक मानव इंटरल्युकिन 4 </td><td> Immunotools </td><td> 11130045 कोई बिल्ली </td></tr><tr><td> एक cyclosporin </td><td> नोवार्टिस </td><td> नहीं एनडीसी 0078-0109-01 बिल्ली </td></tr><tr><td> FBS </td><td> LONZA </td><td> नहीं DE14-802C बिल्ली </td></tr><tr><td> HEPES बफर </td><td> Paa </td><td> कोई S11 001 बिल्ली </td></tr><tr><td> जी 418 सल्फेट </td><td> Paa </td><td> नहीं P02-012 बिल्ली </td></tr><tr><td> / Trypsin EDTA (10x) </td><td> Invitrogen Gibco ® </td><td> 15400-054 रेफरी </td></tr></tbody></table><p class="jove_title"> सी. विविध आपूर्ति: </p><table cellspacing="0" border="1"><tbody><tr><td> <strong>अभिकर्मक</strong> </td><td> <strong>कंपनी</strong> </td><td> <strong>कोई आदेश</strong> </td></tr><tr></tr><tr><td> बाँझ विंदुक युक्तियाँ </td><td> Sarstedt </td><td></td></tr><tr><td> 6 अच्छी तरह से थाली </td><td> NUNC </td><td> 140675 कोई बिल्ली </td></tr><tr><td> 50ml शंक्वाकार ट्यूब </td><td> बी.डी. फाल्कन ™ </td><td> 352070 कोई बिल्ली </td></tr><tr><td> ऊतक संस्कृति फ्लास्क </td><td> SARSTEDT </td><td> 83.1813.002 कोई बिल्ली </td></tr></tbody></table>

generation of human cd40-activated b cells

<p class="jove_content">CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy <sup>1-3</sup>. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) <sup>3</sup>. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded <em>in vitro</em> to very large amounts of highly-pure CD40-B cells (&gt;10<sup>9</sup> cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) <sup>1,4</sup>.</p> 
<p class="jove_content">In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) <sup>5</sup>. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2).</p> 
<p class="jove_content">After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function <sup>1, 4</sup>. CD40-B cells efficiently take up, process and present antigens to T cells <sup>6</sup>. They do not only prime na&#970;ve, but also expand memory T cells <sup>7,8</sup>. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors <sup>9</sup>.</p>

Watch this Scientific Journal Video about Generation of Human CD40-activated B cells Video at JoVE.com

Generation of Human CD40-activated B cells Video (Video) | JoVE

<p class="jove_content">इस वीडियो में हम वर्तमान<em&gt; पूर्व vivo</em&gt; पीढ़ी और मानव CD40 सक्रिय बी कोशिकाओं का विस्तार (CD40 - बी) CD40 ligand और interleukin-4 के साथ उत्तेजना से परिधीय रक्त कोशिकाओं mononuclear (PBMC) से.</p>

मानव CD40 सक्रिय बी कोशिकाओं का सृजन

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

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Generation of Human CD40-activated B cells

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