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"> PBMC的人CD40激活的B细胞产生的协议分为两部分：A部分演示NIH/3T3细胞，将用于板约束的饲养层细胞表达CD40配体的准备。 B部分介绍了各种实际的CD40 - B的文化。 </p><p class="jove_title"> A.制备饲养细胞（N​​IH/3T3 tCD40L） </p><p class="jove_content"> tCD40L NIH/3T3壁小鼠成纤维细胞系，这不应该成为完全融合。因此，这些细胞被劈裂每周两次。超过6周以上的培养，不推荐。 </p><ol><li>从小学文化的老媒体取出，?...

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	<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"> A.媒体的制备： </p><table border="1"cellspacing="0"><tbody><tr><td colspan="2"> <strong>直属细胞野生型中</strong> </td><td colspan="2"> <strong>馈线小区选择培养基</strong> </td></tr><tr><td> <strong>试剂</strong> </td><td> <strong>集中</strong> </td><td> <strong>试剂</strong> </td><td> <strong>集中</strong> </td></tr><tr><td> DMEM - HAM / F12 </td><td></td><td> DMEM - HAM / F12 </td><td></td></tr><tr><td> L -谷氨酰胺</td><td> 365微克/毫升</td><td> L -谷氨酰胺</td><td> 365微克/毫升</td></tr><tr><td>胎牛血清</td><td> 10％ </td><td>胎牛血清</td><td> 10％ </td></tr><tr><td> HEPES </td><td> 10毫米</td><td> HEPES </td><td> 10毫米</td></tr><tr><td>庆大霉素</td><td> 15微克/毫升</td><td>庆大霉素</td><td> 15微克/毫升</td></tr><tr><td></td><td></td><td> G - 418 </td><td> 0.7毫克/毫升</td></tr></table><table cellspacing="0" border="1"><tbody><tr><td colspan="2"> <strong>CD40 - B的清洗介质</strong> </td><td colspan="2"> <strong>CD40 - B的培养基</strong> </td></tr><tr><td> <strong>试剂</strong> </td><td> <strong>集中</strong> </td><td> <strong>试剂</strong> </td><td> <strong>集中</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> L -谷氨酰胺</td><td> 584微克/毫升</td><td> L -谷氨酰胺</td><td> 584微克/毫升</td></tr></tbody><tbody><tr><td> HEPES </td><td> 25毫米</td><td> HEPES </td><td> 25毫米</td></tr></tbody><tbody><tr><td>庆大霉素</td><td> 15微克/毫升</td><td>庆大霉素</td><td> 15微克/毫升</td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> RH转</td><td> 50微克/毫升</td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> RH胰岛素</td><td> 5微克/毫升</td></tr></tbody><tbody><tr><td><br /></td><td><br /></td><td> AB - Humanserum </td><td> 10％ </td></tr></tbody></table><p class="jove_title"> B.其它试剂： </p><table cellspacing="0" border="1"><tbody><tr><td> <strong>试剂</strong> </td><td> <strong>公司</strong> </td><td> <strong>订单号</strong> </td></tr><tr></tr><tr><td> DMEM / HAM的F12 </td><td>临时机场管理局</td><td>猫编号：E15 - 813 </td></tr><tr><td> IMDM </td><td> Invitrogen公司Gibco公司® </td><td>楼盘21980-032 </td></tr><tr><td>贝科的PBS（10X） </td><td>临时机场管理局</td><td>猫没有H15 - 011 </td></tr><tr><td> AB人血清</td><td> Invitrogen公司</td><td> CAT无34005100 </td></tr><tr><td>全息转人类</td><td>西格玛</td><td> CAT没有T0665 </td></tr><tr><td>胰岛素人类</td><td>西格玛</td><td> CAT没有I2643 </td></tr><tr><td> Gencin ® </td><td>三角洲选择</td><td>货号7395800 </td></tr><tr><td>重组人白细胞介素- 4 </td><td> Immunotools </td><td> CAT无11130045 </td></tr><tr><td>环孢素A </td><td>诺华公司</td><td>猫没有NDC 0078-0109-01 </td></tr><tr><td>胎牛血清</td><td>龙沙</td><td>猫没有DE14 - 802C </td></tr><tr><td> HEPES缓冲液</td><td>临时机场管理局</td><td>猫没有S11 - 001 </td></tr><tr><td> G - 418硫酸盐</td><td>临时机场管理局</td><td>猫没有P02 - 012 </td></tr><tr><td>胰蛋白酶/ EDTA（10X） </td><td> Invitrogen公司Gibco公司® </td><td>楼盘15400-054 </td></tr></tbody></table><p class="jove_title"> C.杂项用品： </p><table cellspacing="0" border="1"><tbody><tr><td> <strong>试剂</strong> </td><td> <strong>公司</strong> </td><td> <strong>订单号</strong> </td></tr><tr></tr><tr><td>无菌移液器</td><td> Sarstedt </td><td></td></tr><tr><td> 6孔板</td><td> NUNC </td><td> CAT没有140675 </td></tr><tr><td> 50毫升锥形管</td><td> BD猎鹰™ </td><td> CAT没有352070 </td></tr><tr><td>组织培养瓶</td><td> SARSTEDT </td><td> CAT没有83.1813.002 </td></tr></tbody></table>

generation of human cd40-activated b cells

<p class="jove_content"> CD40激活的B细胞（CD40 - B细胞）已被确定为肿瘤免疫治疗的免疫刺激抗原提呈细胞<sup>（APC）1-3</sup>的替代来源。树突状细胞（DCs），最好的APC，CD40 - B细胞有几个不同的生物和技术性能相比。与区议会，B细胞的MHC和共刺激分子（图1b）的表达增加，表现出强烈的的迁徙能力和目前的T细胞抗原提呈效率，刺激白细胞介素4和CD40配体（CD40L）。然而，在不成熟或成熟的DCS相比，CD40 - B细胞表达完整的淋巴结归巢CD62L，CCR7/CXCR4组成的黑社会，和白细胞功能抗原-1（LFA1，CD11a/CD18），归巢到次级淋巴器官所必需的（图<sup>1a）3。</sup> CD40 - B细胞可以产生而没有从非常小，它可以<em>进一步扩大</em>在体外大量高纯度CD40的B细胞<sup>（&gt;</sup> 10 9细胞每名患者）从健康的捐赠者以及癌症金额外周血困难患者（图1c，d）项<sup>1,4。</sup> </p><p class="jove_content">在这个协议中，我们演示了如何获取充分激活的人PBMC CD40 - B从细胞。细胞培养的关键分子CD40配体，白细胞介素4（IL - 4）和环孢素A（CsA），这是在3-4天养殖周期的补充。 CD40的刺激对于实验室的目的是提供NIH/3T3细胞表达重组人CD40配体（tCD40L <sup>NIH/3T3）5。</sup>为了避免污染与非转染的细胞，表达人CD40配体的转染，定期进行检查（图）。 </p><p class="jove_content"> CD40 - B细胞培养，经过14天，包括95％以上的纯B细胞和CD40 - B细胞超过65天的扩张是没有<sup>任何</sup>损失的<sup>功能：1，4</sup>经常可能。 CD40 - B细胞的有效占用，过程和目前T细胞抗原<sup>6。</sup>他们不只有总理naϊve，而且还扩大记忆性T细胞<sup>7,8。</sup> CD40激活的B细胞可用于研究B细胞的活化，分化和功能。此外，他们代表一个有前途的治疗或预防接种抗肿瘤<sup>9</sup>工具。 </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">在这个视频中，我们目前的<em&gt;体外</em&gt;的产生和扩大人CD40激活的B细胞（CD40 - B）从外周血单个核细胞（PBMC）的刺激与CD40配体和IL - 4。</p>

人CD40激活的B细胞的生成

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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