我们诚恳地感谢克莱门斯Wendtner博士提供tmuCD40配体HeLa细胞株。

协议分为两个部分：第一部分为一代小鼠CD40激活的B细胞（脾mCD40B）一个演示小鼠CD40配体（CD40L）表达HeLa细胞（tmuCD40L HeLa细胞），这将被用作板准备束缚饲养层细胞。 B部分介绍了实际的小鼠CD40 - B的文化。 A.制备饲养细胞（HeLa细胞tmuCD40L） tmuCD40L HeLa细胞是一个附着的人类上皮细胞系，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。 <ol><li>从小学文化的老媒体取出，用无菌吸管和10 mL 1X PBS中洗细胞。吸的PBS洗涤后。 </li><li>添加4毫升1X胰蛋白酶/ EDTA，在一个75厘米的 2瓶10-15分钟，在37 ° C。使用温柔攻分离的细胞。 </li><li>野生型中添加10毫升，并轻轻旋转停止用胰酶消化。 </li><li>转移到50 mL试管细胞悬液，用无菌吸管和旋转265 7分钟XG的细胞。 </li><li>去除上清，重悬细胞沉淀在野生型中的10毫升。计数细胞悬液等分的细胞数，并准备适当数量的细胞50毫升管： <ol><li> 2.5 × 10 6细胞对亚文化</li><li> 0.4 × 10 6细胞/孔照射小鼠CD40 - B细胞培养使用</li><li>其余冻结（如需要）。 </li></ol></li><li>旋转细胞在265 7分钟XG。 </li><li>去除上清。 <ol><li>传代培养：重悬于2.5 × 10 6细胞10毫升，75厘米2细胞培养瓶中的选择培养基（细胞密度为2.5 × 10 5细胞/ mL）孵育细胞在37℃，5％的CO 2 。每周两次分裂的细胞。 </li><li>对于小鼠CD40 - B细胞文化：您需要2.4 × 10 6 6孔板细胞。在野生型中重悬tmuCD40L HeLa细胞密度0.2 × 10 6细胞/ mL，他们在78 Gy的照射。板2毫升细胞悬液，每口井，并在37℃，5％的CO 2。使用B -细胞的刺激，这准备板块tmuCD40L HeLa细胞是贴壁时（至少4个小时：用显微镜检查坚持;不要等到超过24 h开始的B细胞刺激）。 （继续与B） </li></ol></li></ol> B.小鼠CD40 - B细胞培养一，制备脾细胞CD40的刺激（0天）： 请注意：在继续之前确定，饲养层细胞是贴壁。随时添加新的解决办法小鼠白细胞介素- 4，β-巯基乙醇和环孢素A的生长介质，使用前。 <ol><li>剖析的C57BL / 6小鼠（H - 2B单体）的脾和10-20毫升的DMEM洗两次辅以庆大霉素[15μg/mL]一个10毫升吸管尖从一个管到另一个传输。避免使用鼠标的传输污染物（例如头发）。切碎脾细胞通过一个过滤器（100微米）通过，并与其他10毫升的中型冲洗过滤器。降速在室温下为7分钟在265 XG脾，重悬在7ML muCD40 - B细胞中，并Ficoll密度离心分离淋巴细胞。没有在室温下刹车，这样做的5毫升鼠标Pancoll（在室温预热）在15毫升管和离心30分钟XG 450层细胞。 </li><li>绘制关闭最上层，离开淋巴细胞在界面层不受干扰。淋巴细胞层转移到一个新的50 mL试管中，用30毫升，庆大霉素[15μg/mL] DMEM培养液一旦被旋转265 7分钟XG删除其他细胞。弃上清，重悬细胞mCD40 - B的清洗介质在10毫升的。确定细胞的细胞悬液分装数。确定由计数的细胞悬液等分细胞数。 </li><li>离心7分钟在265 XG的细胞所需的金额。对于6孔板5 × 10 6细胞/孔的需要，因此30 × 10 6细胞每盘。 </li><li>取出上清液，重悬mCD40 - B的文化与1 U /白细胞介素4毫升新鲜培养基中的淋巴细胞，在1.25 × 10 6细胞/ mL，生长因子，100微米，β-巯基乙醇和0.63微克/毫升，环孢素A，以防止T细胞的生长（由于浓度是指一毫升培养液！）。 </li><li>取出从6孔板与tmuCD40L HeLa细胞预孵育中的上清。 </li><li>轻轻地添加4毫升的淋巴细胞悬液（1.25 × 10 6细胞/ mL）以及每6孔板。 </li><li>板在37℃，5％的CO 2。 </li&gt; <li>第3天reculture细胞（二.1继续）。 </li></ol>二。 mCD40B细胞传代培养的（第3天，然后每周两次）： <ol><li>收获mCD40B大力移液10毫升到50毫升管的移液器和池他们向上和向下的细胞群。 </li><li>离心7分钟在265 XG，并完全取代mCD40 - B的清洗介质的上清。虽然计数细胞数量的等分，旋转265 XG细胞为7分钟。 mCD40B悬浮细胞mCD40 - B的培养基中，在0.75 × 10 6细胞/ ml的浓度。 </li><li>小鼠白细胞介素4的新鲜的解决方案中添加一个1 U /毫升，100μMβ-巯基乙醇和0.63μg/ mL的中期环孢素A的浓度。 </li><li>取出从6孔板与照射tmuCD40L HeLa细胞预孵育中的上清。 </li><li>轻轻地添加4毫升每6孔板以及mCD40B细胞悬液（0.75 × 10 6细胞/ mL）。 </li><li>孵育板在37℃，5％的CO 2。 </li><li>再次传代培养的细胞，每隔3-4天，以高纯度的小鼠CD40的B细胞激活后的14天总。 </li></ol> C.故障排除：CD40的B细胞，如果不长呢？ <ol><li>你有检查饲养层细胞的CD40的配体表达？ </li><li>板约束馈线用于刺激超过24h以上的细胞？ </li><li>是否有支原体污染吗？ </li><li>是白细胞介素- 4解决方案，为补充新鲜解冻，并有相应的生物活性？ </li><li>是β-巯基乙醇和环孢素A中添加正确的浓度？ </li></ol><img src="/files/ftp_upload/1734/1734fig1a.jpg" alt="图1" /> 图1a。 <img src="/files/ftp_upload/1734/1734fig1b.jpg" alt="图1 B" /> 图1b。 <img src="/files/ftp_upload/1734/1734fig1d.jpg" alt="图1 D" /> 图1d。 <img src="/files/ftp_upload/1734/1734fig2.jpg" alt="图2" /> 图2。

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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> J Clin Invest. 100 (11), 2757-2765 (1997).</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> PLoS ONE. 3, e1464-e1464 (2008).</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=Efficient+generation+of+antigen-specific+cytotoxic+T+cells+using+retrovirally+transduced+CD40-activated+B+cells.">Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells.</a> J Immunol. 169 (4), 2164-2171 (2002).</li><li>Liebig, T. M., Fiedler, A., Zoghi, S., Shimabukuro-Vornhagen, A., 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=Generation+of+human+CD40-activated+B+cells.">Generation of human CD40-activated B cells.</a> J Vis Exp. 32, (2009).</li><li>von Bergwelt-Baildon, M. <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> Blood. 107 (7), 2786-2789 (2006).</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> Cancer Res. 63 (11), 2836-2843 (2003).</li><li>Ahmadi, T., Flies, A., Efebera, Y., Sherr, D. 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L., Grabbe, S., 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=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> Trends Immunol. 25 (12), 659-664 (2004).</li><li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+translocations+are+associated+with+poor+prognosis+in+chronic+lymphocytic+leukemia.">Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.</a> Blood. 107 (2), 742-751 (2006).</li><li>Mason, N. 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实验是按照实验动物饲养的国家和欧洲的指引。在谈到德国的动物福利法，动物是由当局控制的定期的。

本协议的修改是可能的，尤其是关于CD40的刺激和使用的小鼠品系类型的起源，也高产高效代小鼠CD40激活的B细胞。艾哈迈迪等人。表明小鼠CD40的配体转染小鼠成纤维细胞类似的结果。可以肯定地排除在这种异种抗原的背景下演示，但在体内的安全性和毒性的深入研究了在此设置使用人类转染的HeLa细胞的炎症或自身免疫反应的迹象。然而，最好的替代代表与激活和增殖活动在同一时间，这?...

A.媒体的制备： <table border="1"><tbody><tr><td> 直属细胞野生型中 </td><td> 馈线小区选择培养基 </td><td></td></tr><tr><td> 试剂 </td><td> 集中 </td><td> 试剂 </td><td> 集中 </td></tr><tr><td> RPMI 1640培养</td><td></td><td> RPMI 1640培养</td><td></td></tr><tr><td> L -谷氨酰胺</td><td> 300微克/毫升</td><td> L -谷氨酰胺</td><td> 300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>潮霉素B </td><td> 0.2毫克/毫升。 </td></tr></table><table border="1"><tbody><tr><td> mCD40 - B的清洗介质 </td><td> mCD40 - B的培养基 </td></tr><tr><td> 试剂 </td><td> 集中 </td><td> 试剂 </td><td> 集中 </td></tr><tr></tr><tr><td> D - MEM </td><td> </td><td> D - MEM </td><td> </td></tr><tr><td> L -谷氨酰胺</td><td> 580微克/毫升</td><td> L -谷氨酰胺</td><td> 580微克/毫升</td></tr><tr><td>血糖</td><td> 4.5毫克/毫升</td><td>血糖</td><td> 4.5毫克/毫升</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>纪念</td><td> 0.1MM（1X） </td></tr><tr><td> </td><td> </td><td>胎牛血清</td><td> 10％ </td></tr></tbody></table> B.其它试剂： <table border="1"><tr><td> 试剂 </td><td> 公司 </td><td> 订单号 </td></tr><tr><td> RPMI 1640培养</td><td> Invitrogen公司Gibco公司</td><td>楼盘21875 </td></tr><tr><td> D - MEM </td><td> Invitrogen公司Gibco公司</td><td>楼盘41965 </td></tr><tr><td> Dulbeccos PBS（10X） </td><td>临时机场管理局</td><td>猫没有H15 - 011 </td></tr><tr><td> Gencin </td><td>三角洲选择</td><td>货号7395800 </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> MEM NEAA </td><td> Invitrogen公司Gibco公司</td><td>楼盘11140 </td></tr><tr><td>潮霉素B </td><td>临时机场管理局</td><td>猫没有P02 - 015 </td></tr><tr><td>重组小鼠白细胞介素4 </td><td> Immunotools </td><td> CAT无12340045 </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>胰蛋白酶/ EDTA（10X） </td><td> Invitrogen公司Gibco公司</td><td>楼盘15400-054 </td></tr></table> C.杂项用品： <table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 订单号 </td></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>

murine model of cd40-activation of b cells

有研究表明，B细胞CD40的激活提高其抗原提呈能力。当白细胞介素4和CD40配体（CD40L）的刺激下，没有非常大量高纯度的CD40 - B细胞（14天之内的困难，从少量外周血的可扩展人类B细胞&gt; 10<sup&gt; 9</sup&gt;细胞每名患者），从健康的捐赠者以及癌症患者<sup&gt; 1-4</sup&gt;。 CD40 - B细胞表达重要的淋巴结归巢的分子，可以吸引T细胞<em&gt;在体外</em<sup&gt; 5</sup&gt;。此外，他们采取有效，过程和目前的T细胞抗原<sup&gt; 6,7</sup&gt;。 CD40 - B细胞被证明不仅总理天真，但也扩大记忆性T细胞<sup&gt; 8,9</sup&gt;。因此，CD40 -激活的B细胞（CD40 - B细胞）进行了研究，作为一种替代源免疫刺激抗原提呈细胞（APC），基于细胞immunotherapy1，5,10。为了进一步研究是否CD40 - B细胞诱导有效的T细胞在体内的反应和我们建立了小鼠CD40激活的B细胞产生的细胞培养系统研究的基础机制。使用脾细胞或纯化C57BL / 6小鼠的B细胞CD40的激活，确定了最佳工艺条件如下：开始的C57BL / 6小鼠淋巴细胞（单体型H - 2B）的脾细胞密度梯度离心法和HeLa细胞共同培养纯化重组鼠CD40配体（tmuCD40L的HeLa细胞表达）<sup&gt; 11</sup&gt;。细胞是再培养每隔3-4天，和关键部件，如CD40L，白细胞介素- 4 -巯基乙醇和环孢素A的补充。在这个协议中，我们演示了如何获得类似的APC -表型的人CD40 B细胞（图1A，B）完全激活的小鼠的CD40 - B细胞（mCD40B）。 CD40的刺激会导致快速的生长和细胞培养在14天的高纯度（&gt; 90％），CD19 + B细胞的扩张（图1C，D）​​。为了避免污染与非转染的细胞，小鼠CD40的配体的转染表达经常被控制（图2）。小鼠CD40激活的B细胞可用于研究B细胞的活化和分化，以及调查他们的潜能作为APC，在体内和体外的功能。此外，他们代表了建立抗肿瘤治疗或预防接种有前途的工具，将有助于回答问题，这种方法的安全性和免疫原性<sup&gt; 12</sup&gt;。

Watch this Scientific Journal Video about Murine Model of CD40-activation of B cells at JoVE.com

Murine Model of CD40-activation of B cells

在这个视频中，我们展示的CD40从C57BL / 6小鼠的脾细胞，它可以用来作为模型抗原提呈细胞（APC）的研究免疫诱导小鼠的B细胞的活化和扩大的过程。

激活的B细胞CD40的小鼠模型

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

murine-model-of-cd40-activation-of-b-cells

Research

JoVE Journal

Immunology and Infection

12.6K 次观看.  University Hospital of Cologne. 在这个视频中，我们展示的CD40从C57BL / 6小鼠的脾细胞，它可以用来作为模型抗原提呈细胞（APC）的研究免疫诱导小鼠的B细胞的活化和扩大的过程。

视频: Murine Model of CD40-activation of B cells

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

纯净小鼠的B细胞的诱导和评估类开关重组

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

科研

免疫与感染

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

重组逆转录病毒的生产和B细胞的感染

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

对小鼠诺沃克类病毒的空斑法

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

检测真正的IgE的表达小鼠B​​系细胞

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

检测和罕见富集抗原特异性B细胞的表型和功能分析

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

人正常记忆 b 细胞与长寿浆细胞体外分化模型

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...