我们非常感谢有益的讨论马丁实验室。本刊物是由加拿大健康研究协会（澳门币89783）的赠款支持。我是支持由一个加拿大研究主席II级奖。

第一步：通过磁富集脾B细胞分离<ol><li>实验小鼠须年满8-12周，以确保充分成熟的免疫系统。 </li><li>颈椎脱位安乐死鼠标和70％的乙醇浸泡。通过左季肋部，腹部皮肤和肌肉的切口手术去除脾切成磷酸三大块缓冲液（PBS）。确保所有的工具和试剂是无菌的。 </li><li>使用1 mL注射器柱塞橡胶年底，轻轻捣烂脾细胞过滤器通过70微米到PBS。请务必使用一个推的议案，而不是磨议案的研磨可能会导致较大的（即增殖）细胞破裂。 </li><li>离心5分钟不超过350克的细胞，并在PBS悬浮颗粒。使用血球计数悬浮细胞。 </li><li>准备用5％的5毫升无菌聚苯乙烯管的正常大鼠血清与第（2毫升每管最大音量）暂停1 × 10 8个细胞/毫升的PBS 。 </li><li>加入50μLEasySep负鼠标选择B细胞每毫升细胞富集的鸡尾酒。移液器的混合物，帽管，并在15分钟的冰箱。 </li><li>每毫升细胞，加入100μLEasySep生物素选择鸡尾酒。移液器的混合物，帽管，并在15分钟的冰箱。 </li><li>每毫升细胞，加入100μLEasySep磁性纳米粒子（确保纳米粒子充分混合的解决方案是同质）。移液器的混合物，帽管，并在5分钟的冰箱。 </li><li>顶部的解决方案，并在5分钟EasySep磁铁用PBS地方，以2.5毫升。反转的磁铁和管，并迅速倒入一个新的聚苯乙烯管。不要动摇管负选定的单元格将磁约束管壁。 </li><li>为了进一步提高纯度的细胞悬液，管重新插入到一个额外的5分钟的磁铁，并灌入新管。这可能会降低整体细胞恢复。 </li><li>流动的B细胞表面标志的流式细胞仪分析，B细胞纯度可评估。浓缩之后，我们不断地看到纯度&gt; 95％B220阳性细胞。 </li></ol>第二步：CFSE细胞染色<ol><li>在温暖的PBS重悬的分离出的细胞，在终浓度为1 × 10 6细胞每毫升0.1％牛血清白蛋白补充。 </li><li>准备一个5mm的原液CFSE（稀释后在无菌二甲基亚砜），并为每毫升细胞在试管中添加2μL。 10μm的最终浓度是初级B细胞染色的最佳选择。 </li><li>在黑暗中10分钟在37 ° C（注：CFSE对光敏感，应保持在黑暗中的可能时）。 </li><li>淬火牛小牛血清等体积加入你的细胞，并在冰上孵育5分钟的污点。 </li><li>洗细胞培养基补足管（见配方步骤三）在350离心G.请注意，至少5倍的量相当于媒体必须加入抵制牛小牛血清的粘度和细胞产生沉淀。 </li><li>清洗细胞两次培养基。现正准备为刺激细胞。 </li></ol>第三步：LPS刺激细胞<ol><li>准备在体外 B细胞培养液中，通过补充10％胎牛血清和50μMβ-巯基乙醇/的RPMI 1640 。 </li><li>准备加入LPS的终浓度为50μg/mL刺激中等。分装125μL刺激介质以及每一个平底96孔组织培养板。 </li><li>准备您的CFSE染色的B细胞培养液中无LPS终浓度在3.2 × 10 6细胞/ mL 。加入125μL的细胞悬液含有刺激培养基轻轻吹打混合井。 </li><li>现在每口井包含在最后的LPS的浓度为25微克/毫升的4 × 10 5细胞。这提供了细胞增殖和类别转换的最佳水平，虽然根据需要更改这些值可能。 </li><li>细胞在37℃，5％CO 2 72至96小时。 48小时后，大量增殖的B细胞群将在显微镜下清晰可见。 </li></ol>第四步：流量级交换的流式细胞仪分析<ol><li>当您准备好来看待级交换，删除你的细胞，从他​​们的文化条件和1毫​​升染色缓冲液（2％牛小牛血清的PBS）洗两次。 </li><li>为了防止你的细胞，颗粒细胞的非特异性抗体染色，纺纱350克和incuba特在5％的正常小鼠血清和FcBlock per106细胞在冰上15分钟1μg染色缓冲100μL。细胞阻塞，应在冰上进行。 </li><li>不洗你的细胞，每一个荧光标记的抗小鼠IgG3抗体10 6细胞添加1μg，使细胞染色30分钟，在冰上。 </li><li> 200-300染色缓冲液中的悬浮离心洗涤细胞两次。这些细胞是目前评估流式细胞仪准备就绪。 </li><li> CFSE是最佳的兴奋在492 nm处（氩离子激光器），并在517 nm处发出。 IgG3抗体激发和发射光谱是取决于它的共轭荧光染料。 </li></ol>代表性的成果磁性富集，细胞悬液应该看起来像一个同质人口难以区别小圆形细胞（图1A）。刺激后48-72小时，扩大增殖细胞的隔离集群将清晰可见（图1B）。会出现一个大比例的非增殖细胞和颗粒，因为它们发生凋亡。 CSR通常可检测刺激后，在72和96小时之间的最高水平之前，大部分细胞开始死亡7。在这一阶段，细胞发生了细胞分裂交换出现在后来的女儿细胞群的细胞数量。图2可以看出，在代表流动CFSE稀释和表面的LPS刺激后96小时IgG3表达的流式细胞仪积。 表1。抗体同种型开关鸡尾酒。注：兴奋剂浓度值是唯一的建议。滴定法可能是必要的的。 <table border="1"><tbody><tr><td>期望中的同型</td><td>刺激鸡尾酒</td></tr><tr><td> IgG1and的IgE </td><td> LPS（25μg/mL）和IL - 4（10毫微克/毫升） </td></tr><tr><td> IgG1and的IgE </td><td>抗CD40（10μg/mL）和IL - 4（10毫微克/毫升） </td></tr><tr><td> IgG2a </td><td> LPS（25μg/mL）和干扰素-γ（10毫微克/毫升） </td></tr><tr><td> IgG2b和IgG3 </td><td> LPS（25μg/mL） </td></tr><tr><td> IGA </td><td> LPS（25μg/mL），转化生长因子-β（2毫微克/毫升）和IL - 5（1.5毫微克/毫升） </td></tr></tbody></table><img src="/files/ftp_upload/2130/2130fig1.jpg" alt="图1" /> 图1。脾B细胞的分离和LPS刺激。 （一）前防磁丰富LPS刺激脾B细胞。 （二）与25微克/毫升的LPS刺激后48小时。增殖细胞被视为爆破和细胞凋亡的背景集群。 <img src="/files/ftp_upload/2130/2130fig2.jpg" alt="图2" /> 图2。扩散和类开关重组后96小时。 （一）CFSE稀释对LPS刺激细胞后96小时在文化。每个独立的峰代表稀释CFSE荧光对应一个独立的细胞分裂。 （二）IgG3表达一个IgG3阳性人群中，细胞分裂的几个回合后，出现。

<ol>
	<li>Hodgkin, P. D., Lee, J. H., Lyons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+differentiation+and+isotype+switching+is+related+to+division+cycle+number.">B cell differentiation and isotype switching is related to division cycle number.</a> J Exp Med. 184, 277-281 (1996).</li><li>McCall, M. N., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+recombination+and+germ-line+transcription+are+division-regulated+events+in+B+lymphocytes.">Switch recombination and germ-line transcription are division-regulated events in B lymphocytes.</a> Biochim Biophys Acta. 1447, 43-50 (1999).</li><li>Manis, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+links+DNA+damage-response+pathways+to+immunoglobulin+heavy+chain+class-switch+recombination.">53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination.</a> Nat Immunol. 5, 481-487 (2004).</li><li>de Yebenes, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-181b+negatively+regulates+activation-induced+cytidine+deaminase+in+B+cells.">miR-181b negatively regulates activation-induced cytidine deaminase in B cells.</a> J Exp Med. 205, 2199-2206 (2008).</li><li>McBride, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+class+switch+recombination+and+somatic+mutation+by+AID+phosphorylation.">Regulation of class switch recombination and somatic mutation by AID phosphorylation.</a> J Exp Med. 205, 2585-2594 (2008).</li><li>Ramachandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNF8/RNF168+ubiquitin+ligase+cascade+facilitates+class+switch+recombination.">The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination.</a> Proc Natl Acad Sci U S A. 107, 809-8014 (2010).</li><li>Zaheen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+constrains+germinal+center+size+by+rendering+B+cells+susceptible+to+apoptosis.">AID constrains germinal center size by rendering B cells susceptible to apoptosis.</a> Blood. 114, 547-554 (2009).</li><li>Chaudhuri, J., Alt, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class-switch+recombination:+interplay+of+transcription,+DNA+deamination+and+DNA+repair.">Class-switch recombination: interplay of transcription, DNA deamination and DNA repair.</a> Nat Rev Immunol. 4, 541-552 (2004).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli+that+enhance+IgA+class+switching+increase+histone+3+acetylation+at+S+alpha,+but+poorly+stimulate+sequential+switching+from+IgG2b.">Stimuli that enhance IgA class switching increase histone 3 acetylation at S alpha, but poorly stimulate sequential switching from IgG2b.</a> Eur J Immunol. 37, 240-251 (2007).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+class+switching+differs+among+SJL,+C57BL/6+and+129+mice.">Antibody class switching differs among SJL, C57BL/6 and 129 mice.</a> Int Immunol. 19, 545-556 (2007).</li></ol>

上文所述的协议提供了一个标准的检测，以分析在初级小鼠的B细胞级交换援助的表达和功能。该协议使用细菌脂多糖诱导从IgM的切换到IgG3，虽然刺激媒体的修改，可以切换到其他的亚型（总结在表1 8，9 ）诱导。 我们注意到，对于未知的原因，小牛血清能影响类切换在体外观察到的水平。至关重要的是，同一批号的所有CSR实验，以确保实验的一致性。屏幕面板的胎牛血清来源，在体外交换速率达到最佳，这也可能是有益的。小鼠品系也可以看到在体外 10级交换的影响程度。根据检测问题，不良小鼠模型，如SJL鼠标切换，必须有足够的回交C57BL / 6背景，以达到明显的切换率。 步我可以省略，如果不希望一个纯B细胞培养。刺激脾细胞的混合物仍然会诱发类开关，虽然它可能会导致人口较多的凋亡非B细胞。此外，随后的流式细胞仪分析，必须包括一个既定的B细胞标记（如B220，CD19 +）。在本议定书中所用纯化试剂盒提供的干细胞技术（见试剂表）。根据制造商的指示，也可用于使用负的浓缩过程的备用套件。 CFSE荧光可以很激烈​​，因此每次实验需要一个适当的补偿控制流式细胞仪分析。此外，CFSE会慢慢泄漏，导致减少随着时间的推移的整体荧光的细胞。基于这些原因，建议，所有的实验，包括未刺激CFSE染色的细胞群。在很短的时间内，这些细胞将保持静态的文化，因此可被用于作为补偿CFSE控制和识别的非增殖的细胞群。

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		<th>Catalogue Number</th>
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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
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<td>EasySep Mouse B Cell Enrichment Kit</td>
<td>&nbsp;</td>
<td>Stemcell Technologies</td>
<td>19754</td>
<td>&nbsp;</td>
<tr>
<td>Polystyrene tubes</td>
<td>&nbsp;</td>
<td>Becton Dickinson Falcon</td>
<td>352058</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>CellTrace CFSE Cell Proliferation Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C34554</td>
<td>&nbsp;</td>
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<td>Bovine Calf Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30072.03</td>
<td>&nbsp;</td>
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<td>RPMI 1640 Culture Medium</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
<td>&nbsp;</td>
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<td>Lipopolysaccharides from Escherichia coli</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>L6529</td>
<td>&nbsp;</td>
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<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21985</td>
<td>&nbsp;</td>
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<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30396.03</td>
<td>&nbsp;</td>
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<td>Normal Mouse Serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>011-000</td>
<td>&nbsp;</td>
<tr>
<td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553141</td>
<td>&nbsp;</td>
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<td>Rat Anti-Mouse IgG3</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553401</td>
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induction and assessment of class switch recombination in purified murine b cells

体液免疫是维持B细胞通过分泌的抗体介导的免疫系统的分支。 B细胞活化后，免疫球蛋白轨迹经历了一系列基因修饰，改变的结合能力和分泌的抗体效应功能。这个过程是由一个基因重组事件被称为类开关重组（CSR），其中默认IgM抗体亚型的IgG，IgA或IgE的替代突出。每个亚型具有不同的效应功能，从而使企业社会责任的关键，以维护免疫力的。 酶的活化诱导胞苷脱氨酶（AID）是介导的免疫球蛋白基因座多样化。详细地描述这个过程，是一个原理的视频可在网上（http://video.med.utoronto.ca/videoprojects/immunology/aam.html）。援助活动和企业社会责任的途径通常在B细胞功能和小鼠的体液免疫的评估研究。在本报告所述的协议提出了一种从小鼠脾脏和随后的刺激与细菌脂多糖（LPS）诱导级交换IgG3（其他抗体亚型见表1）的B细胞的分离方法。此外，荧光细胞染色染料羧基荧光素琥珀酰亚胺酯（CFSE）是用来监视刺激的细胞，细胞分裂的过程中的关键同型开关1，2 。 监管援助和企业社会责任的发生机制，目前仍不清楚， 从而在体外类开关检测提供了一种可靠的方法，测试在不同的小鼠模型，这些进程。这些检测先前已在基因缺乏使用基因敲除小鼠3的情况下使用。此外，B细胞在体外开关之前，病毒转导来调节基因表达的RNA敲除或转基因表达的4-6。这些类型的实验数据，影响我们理解的援助活动，分辨率CSR的反应，并在小鼠的抗体介导的免疫力。

Watch this Scientific Journal Video about Induction and Assessment of Class Switch Recombination in Purified Murine B Cells at JoVE.com

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

抗原暴露后，激活的B细胞亚群接受类开关重组（CSR）的一个过程，产生不同的效应功能的抗体亚型。在本报告所述的协议解释CSR如何可诱发和分析<em&gt;在体外</em&gt;为研究B细胞功能的目的。

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 ...

induction-assessment-class-switch-recombination-purified-murine-b

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.1K 次观看.  University of Toronto. 抗原暴露后，激活的B细胞亚群接受类开关重组（CSR）的一个过程，产生不同的效应功能的抗体亚型。在本报告所述的协议解释CSR如何可诱发和分析在体外为研究B细胞功能的目的。

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

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 ...

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

在纯化造血干/祖细胞识别DNA突变

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

人骨髓间充质干细胞（干细胞）的免疫调节性能评估

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

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 ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in 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 diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

粒子结合Bet v 1的变应原PM10的流式细胞分析

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 ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

HDAC 抑制剂介导的 B 细胞 rna 和基因的基因组分析--诱导进行类开关 DNA 重组和血浆细胞分化

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

体外和活体评估移植受体的 T、B 和髓细胞抑制活性和体液反应

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...