Name: induction and assessment of class switch recombination in purified murine b cells
Uploaded: 2010-08-13T17:00:00
Description: Watch this Scientific Journal Video about Induction and Assessment of Class Switch Recombination in Purified Murine B Cells at JoVE.com

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...

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	<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?...

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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</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>
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<td>Polystyrene tubes</td>
<td>&nbsp;</td>
<td>Becton Dickinson Falcon</td>
<td>352058</td>
<td>&nbsp;</td>
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<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
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<td>CellTrace CFSE Cell Proliferation Kit</td>
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<td>Invitrogen</td>
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<td>Bovine Calf Serum</td>
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<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>
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<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>
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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。

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