这项工作是由美国国立卫生研究院以R01 NS071039-01A1研究资助支持。

1。从正常中枢神经系统小胶质细胞的分离小胶质细胞的分离以前修改3。 <ol><li>解剖和血流准备工具：剪刀，镊子，10毫升注射器27克针，15毫升Dounce匀浆（铁氟龙/玻璃），40微米尼龙细胞过滤器，40％和70％的Percoll方案，1×PBS。 </li><li>安乐死小鼠CO 2的相机快窒息（缺氧），并立即转移，手术操作和灌注的实验台。 </li><li>执行手术操作（用剪刀，镊子，70％的乙醇作为防腐剂）：打开腹腔，切开膈肌，打开胸腔，并用剪刀剪断右心房。 </li><li>左心室注入10毫升的注射器和施加压力，进行心内灌注全身。通过血液循环，在缓慢而稳定的PA 8-10毫升的1X PBSCE。 </li><li>组中的所有小鼠（通常5-10小鼠）重复步骤1.2-1.4。灌注后继续，直到所有的老鼠都灌注冰鼠标。 </li><li>所有小鼠灌注后，解剖大脑和脊髓。 （可选：研究在大脑的不同区域的miRNA的表达，如小脑，海马等为5-10小鼠的同质化和池一起剖析这些具体领域）。 </li><li>使用15毫升Dounce均质均质的大脑和脊髓。放置一个大脑和一个脊髓匀浆和添加5毫升的1X PBS。执行10-20温柔的罢工;​​ 50 ML-锥形管和证书在2000年上一个大的离心5-7分钟的转速通过细胞过滤成匀浆。 </li><li>弃上清，重悬2-3 5-7毫升70％的Percoll和覆盖7毫升40％的Percoll小鼠脑/脊髓匀浆。 30分钟旋转2000转（无刹车）。 </li><li>放弃70％的顶部受损细胞/髓鞘碎片，并收集单核细胞在40％/ 70％的界面。至少在2000年5-7分钟转速1:1 1X PBS和自旋稀释收集细胞。在0.5毫升的PBS重悬细胞和转移到1.7毫升ependorf管。典型的产量是500×10单核细胞每鼠标，这将使准备从2-3小鼠的大脑和脊髓的细胞小胶质细胞的数量〜1×10 6 0.5毫升3。 </li></ol> 2。控制小胶质细胞的纯度流式细胞仪抗体染色进行修改7,8与先前描述。 <ol><li>准备缓冲流式细胞仪（1X与2％FBS，PBS），FCR封闭抗体，抗CD11b -体育，抗CD45 - APC-Cy7抗体在PBS，1％多聚甲醛。 </li><li>采取步骤1.9中的细胞从50μl和他们转移到新的1.7毫升的Eppendorf管中，并添加350流式细胞仪缓冲液。 </li><li>加入5-10μl和抗Fc受体抗体孵育10-15分钟在冰上。 </li><li>分裂成两个Eppendorf管细胞。一管加入1μl的抗CD11b - PE和2μl抗CD45-APC-Cy7抗体，并在冰上孵育10-15分钟。第二条管道将被用来作为阴性对照抗体染色。抗体孵化后添加1毫升流式细胞仪缓冲管和自旋为5400 rpm离心5分钟。经过纺纱，解决他们的细胞重新悬浮于400μL的1％多聚甲醛PBS和涡旋。 </li><li>流式细胞仪使用补偿控制BD compubeads（BD Biosciences公司）是8（LSR的二，BD Biosciences公司）执行流仪分析。 如图好和坏的筹备工作的例子。 1A，B。 </li></ol> 3。在小鼠模型的神经发炎红肿中枢神经系统小胶质细胞和外周血巨噬细胞的分离正如先前所描述的实验性自身免疫性脑炎（EAE）的诱导在我们的文件第7;流式细胞仪分选进行类似出版协议8。 <ol><li> 8-12周龄C57BL / 6小鼠在4毫克/毫升完全弗氏佐剂（CFA）5-10组150毫克MOG 35-55肽诱导皮下免疫脑脊髓炎。百日咳毒素应给予天零和免疫后第二天，IP（150毫微克/鼠标）。小鼠观察疾病的免疫后10天开始的迹象，并取得了疾病的严重程度从0-5的数字规模如下：0）无病; 1）弱尾或步行摇晃; 2）后肢麻痹; 3）后肢瘫痪; 4）后肢和前肢瘫痪; 5）因人道原因死亡或安乐死。用于疾病具有典型的得分从1.5到2.5不等的实验小鼠在疾病高峰期（D21后免疫）。 </li><li>从步骤1.1-1.4 5-10小鼠中枢神经系统中分离单核细胞和染色步骤2.1-2.4抗CD11b和抗CD45抗体的细胞。在步骤2.4不修复细胞，而不是他们悬浮在400μl1X PBS代替1％多聚甲醛的。 </li><li> FACSAria人口的CD11b + CD45的低的小胶质细胞和CD11b + CD45的喜细胞（〜80％的人是周边的巨噬细胞和〜20％，其中被激活小胶质细胞3;在未来，我们将把这些细胞是通过细胞分选“巨噬细胞“）。用于样品收集1.7毫升ependorf管的。 300 1X与10％FBS的PBS液应加入到收集管，以防止排序的细胞损伤。 如图三个群体小胶质细胞，巨噬细胞和淋巴细胞的例子。与适当的排序门设置， 如图2A。 2B。典型的排序纯度均在98-100％，重新分析排序人口的小胶质细胞（ 图2c）和巨噬细胞（ 图2d）确定。典型的产量是3 X10的小胶质细胞和20-503 50-100 X10从五鼠组分离的巨噬细胞。 </li></ol> 4。 RNA提取 RNA会被孤立使用mirVana试剂盒，根据制造商的指示修改。 <ol><li>旋转小胶质细胞从正常的中枢神经系统从1.9步排序的子集或小胶质细胞和巨噬细胞在小离心5-7分钟5400转3.3 1.7毫升的Eppendorf管中一步，小心弃上清和细胞颗粒干冰冻结。继续加强4.2或冷冻细胞颗粒转移到-70℃，它可以储存2-3个月。 </li><li>转让定期冰解冻冷冻细胞颗粒。 mirVana套件新增300μl裂解液的轻轻混合pipeting 5-7倍。 </li><li>添加mirVana套件，涡旋30秒30的μl匀浆添加剂的混合，保持10分钟冰</li><li>加入氯仿300μL酸Pehnol：从mirVana套件，涡旋30秒，自旋5米在小离心机上10000转。 </li><li>仔细考虑上层水相（300μL），并转移到另一个1.7毫升的Eppendorf管中，用375μl100％乙醇的混合，转移到过滤从mirVana试剂盒和自旋为1分钟10,000转的墨盒。 </li><li>丢弃流通过缓冲区，添加700μl洗涤液我从mirVana套件，并在10,000 rpm的自旋为1分钟的。 </li><li> 500洗涤液微升II从mirVana套件洗超过两次，终于洗脱RNA 60μL预热无核酸酶水从墨盒。 </li><li>的RNA的数量和质量进行评估，使用的NanoDrop分光光度计。 RNA的浓度应为5至20毫微克/毫升和外径λ260/λ2801.7-2.0范围内的比例。如果RNA浓度低于3 ng / ml的和260λ/280λ外径大于1.6低，样品将被丢弃。 RNA样品可存放于-70℃为6-12个月。 </li><li>为进一步估计RNA，样品AR质量é分析，15％，简称TBE-尿素凝胶（Life Technologies公司）使用凝胶电泳。 如图的RNA制剂质量好和坏的典型例子。 3a和图3b分别。 </li></ol> 5。在小胶质细胞和巨噬细胞的miRNA检测分析miRNA的表达，实时定量逆转录聚合酶链反应（QRT-PCR）分析进行了引物和荧光探针，为特定的microRNA的兴趣和广泛表达的短的RNA（如的snoRNA-55，snoRNA的使用TaqMan分析-135或U6）正常化。特别是集人类或小鼠的miRNA的引物和荧光探针，可以购买生命科技公司，在这里我们将用miR-124的snoRNA的利益和正常化的一个例子-55的miRNA的一个例子。 <ol><li>进行逆转录反应，使从RNA样品使用TaqMan反转录试剂盒基因： </li></ol><table border="1"><tbody><tr><td> 项目 </td><td> 每个样品量（μl） </td></tr><tr><td> 5X RT引物 </td><td> 1.00 </td></tr><tr><td> RNA样品 </td><td> 1.67 </td></tr><tr><td> RT酶混合物 </td><td></td></tr><tr><td> 25毫米（100毫米总）dNTPs浓度</td><td> 0.050 </td></tr><tr><td> MultiScribe逆转录） </td><td> 0.333 </td></tr><tr><td> 10X逆转录缓冲器</td><td> 0.500 </td></tr><tr><td> AB酶抑制剂</td><td> 0.063 </td></tr><tr><td>无核酸酶水</td><td> 1.388 </td></tr><tr><td> 总 </td><td> 5.00 </td></tr></tbody></table>稀释浓度为3 ng /μl的RNA样品。准备逆转录酶混合物和3.33μL的PCR管和添加1.67μLRNA样品。在16°C孵育PCR仪（BioRad公司）30分钟，42℃30分钟，85℃为5分钟，在4°C间搁置。 <ol start="2"><li>执行实时PCR反应使用TaqMan PCR混合物： </li></ol><table border="1"><tbody><tr><td> 项目 </td><td> 每个样品量（μl） </td></tr><tr><td> RT产品 </td><td> 1.0 </td></tr><tr><td> 2X的TaqMan预混 </td><td> 5.0 </td></tr><tr><td> 5X探头 </td><td> 2.0 </td></tr><tr><td> 无核酸酶水 </td><td> 2.0 </td></tr><tr><td> 总 </td><td> 10.0 </td></tr></tbody></table>使用384以及清晰的光学反应，每个反应和实时PCR仪AB 7900羟色胺板（生命技术）。循环条件：95°C为10分钟，95℃，5秒，60°，60秒D]×40次。 <ol start="3"><li>氟量的典型曲线rescently标记的特异性PCR产物的snoRNA-55和miR-124（Y轴），小胶质细胞和骨髓巨噬细胞（BMDMs）与周期数（X轴）分离RNA样品（每个样品一式两份） 如图。 4A，B。 </li></ol> 6。正常化和数据分析如前所述7,9使用snoRNA的初始金额的RNA正常化55ΔΔCT法计算miRNA的利息（MIR-124）的相对表达水平。 <ol><li>计算的相对水平的miR-124表达对小胶质细胞和BMDMs例如表一所示。 </li><li>作为第一步，Ct值的snoRNA-55和miR-124被确定为小胶质细胞和BMDMs QRT-PCR曲线（ 图4;表一 ，列的snoRNA-55和miR-124）。 </li><li>作为第二步，ΔCT值每个样品重复Ct的减法计算的miR-124（实验）（ 表，列：CT（规范），CT（EXP），和ΔCT）从CT的snoRNA-55（正常化）。 </li><li>作为第三步。 ΔΔCT值计算减法ΔCT的参考样本（ΔCT（价））从其他样本（ 表一栏：ΔΔCT）ΔCT值。定义一个样本作为参考，将被定义为1.0的这个样品的miRNA表达的相对水平。相比其他所有样品将这个参考样本。在我们的例子中，我们定义为小胶质细胞的第一个样本为1.0 miR-124的水平，因此ΔCT（REF）= 0.49（ 表一 ，第一行，用红色标出）。 </li><li>最后，相对表达水平的计算公式为2-ΔΔCT（ 表一栏：MIR-124éxPression可）。 </li><li>所有定量RT-PCR的进行，在一式三份或重复，相对表达水平为平均±标准差（SD）的一式三份（ 图5A，B）或两个边重复边图所示。 5C。 </li></ol> 7。代表结果实时PCR和miR-124的CT值（microRNA的利益）的snoRNA-55（正常化）以及相对的miR-124表达水平的典型曲线图所示。 4和表。在我们的实验中，我们作为一个正常化的控制snoRNA的55。正如我们上面提到的，无处不在的RNA，可也用于snoRNA的135和U6如正常化。 如图表达的miR-124的成人小胶质细胞与骨髓衍生的巨噬细胞（BMDMs）的例子。 5A（BMDMs增长的presencE为M-CSF的描述7）。 图。 5B，我们比较在人口排序的CD45 低的小胶质细胞与CD45的喜巨噬细胞的miR-124的表达。我们在这两个实验证明，小胶质细胞是从其他的巨噬细胞表达的miR-124的更高水平的不同。在未来可以进行类似的实验，小胶质细胞在体内或体外激活动力学。 如图表达的miR-124的小胶质细胞的C57BL / 6小鼠在不同发展阶段的中枢神经系统中分离。 5C。这一数据表明，在发展快递低miR-124的水平，这与他们的活化型7相关的早期阶段，小胶质细胞。可以进行类似的分析，研究小胶质细胞分离的中枢神经系统的不同领域特别是在小胶质细胞miRNA的表达比较正常，如中枢神经系统的功能：小脑的皮纳尔线，海马等。 <img alt="图1" src="/files/ftp_upload/4097/4097fig1.jpg" /> 图1。好的和坏的小胶质细胞通过流式细胞仪检测的准备的例子。在“好”的小胶质细胞脱离正常的中枢神经系统的情况下，代表95-98％的细胞CD11b的2-5％的CD11b + CD45 低 microgla的+ CD45的喜血管周围巨噬细胞。应该小于1％的CD11b -您好 CD45的淋巴细胞或细胞CD11b - CD45的-星形胶质细胞，少突胶质细胞或细胞碎片（ 一 ）。 （b）在18％的污染细胞CD11b - CD45的-细胞是（B，左下象限），建议Percoll梯度分离不足，这里的“坏”的筹备情况。在做好准备的情况下，有95-98％的细胞应该代表细胞CD11b + CD45低，小胶质细胞（A，左上象限），应代表所有细胞的2-5％细胞CD11b + CD45的喜 （血管周围巨噬细胞，右上象限），并应代表所有细胞的不到1％CD11b的负面污染细胞（ 一 ，左下象限）。在“坏的准备”的情况下，重大污染的CD11b - CD45的-细胞或细胞碎片（星形胶质细胞，髓鞘颗粒等）（B，左下象限），这使得细胞RNA分离，并进一步不适宜做准备分析。此外，CD11b的- CD45的喜细胞可能在“坏的准备”的情况下也存在（B，右下象限），说明由于不足灌注血液淋巴细胞的污染。 <img alt="图2" src="/files/ftp_upload/4097/4097fig2.jpg" /> 图2。流量仪analysi和小胶质细胞和外周血巨噬细胞数量排序（ 一 ）在发炎反应的细胞群体是存在于中枢神经系统病变的中枢神经系统细胞CD11b + CD45 低 （ 小胶质细胞 ）， 细胞CD11b + CD45的喜 （ 巨噬细胞 ）， 和 CD11b - CD45; 您好 （ 淋巴细胞 ），分别显示在左上角，右上角和右下角的象限。 + CD45的喜巨噬细胞CD11b + CD45的低小胶质细胞和CD11b排序人口盖茨（b）和重新分析排序种群的纯度（C，D）所示。实现对活细胞的初步门，FSC / SSC参数。 <img alt="图3" src="/files/ftp_upload/4097/4097fig3.jpg" /> 图3。 “好”与“坏”qualit范例在质量良好的情况下，RNA的RNA分离和小胶质细胞凝胶电泳分析IES。梯子和核糖体RNA是显而易见的（ 一 ）。在质量差，涂抹，低分子量降解产物是显而易见的（B）。 <img alt="图4" src="/files/ftp_upload/4097/4097fig4.jpg" /> 图4。实时定量RT-PCR扩增曲线荧光标记的miR-124的snoRNA-55的PCR产物。的snoRNA-55（A）和miR-124（B）曲线显示小胶质细胞和骨髓衍生的巨噬细胞（BMDMs）。实验是在重复。 Ct值由下部的线性范围QRT-PCR的基线的交点决定的曲线在（b）所示。 <a href="http://www.jove.com/files/ftp_upload/4097/4097fig4large.jpg" target="_blank">点击这里查看大图</a> 。 <table border="1"><tbody><tr&gt; <td></td><td> snoRNA的-55 </td><td> miR-124的 </td><td> DCT = CT（EXP）-CT（范） </td><td> DDCT = DCT-DCT（REF） </td><td> 表达的相对水平= 2-DDCT </td></tr><tr><td></td><td> CT（范） </td><td> CT（EXP） </td><td> DCT </td><td> DDCT </td><td> 的miR-124表达 </td></tr><tr><td> 小胶质细胞从正常的中枢神经系统 </td><td> 24.082 </td><td> 24.572 </td><td> 0.49（参考） </td><td> 0 </td><td> 1.0 </td></tr><tr><td></td><td> 23.766 </td><td> 24.291 </td><td> 0.525 </td><td> 0.035 </td><td> 1.02 </td></tr><tr><td> 骨髓衍生的巨噬细胞（BMDMs） </td><td> 26.879 </td><td> 320.231 </td><td> 5.352 </td><td> 5.317 </td><td> 0.025 </td></tr><tr><td></td><td> 26.526 </td><td> 32.078 </td><td> 5.552 </td><td> 5.517 </td><td> 0.022 </td></tr></tbody></table> 相对水平的miR-124表达在小胶质细胞与骨髓巨噬细胞C T值的miR-124（microRNA的利益）和snoRNA的55（控制正常化）根据表一计算。 <img alt="图5" src="/files/ftp_upload/4097/4097fig5.jpg" /> 图5。分析的miR-124在小胶质细胞和巨噬细胞中的表达。比较小胶质细胞的miR-124表达从正常的中枢神经系统对骨髓巨噬细胞（BMDMs）在（a）所示。在小胶质细胞与神经发炎小鼠中枢神经系统分离外周血巨噬细胞中的miR-124的比较（b）中所示。在变化（c）中所示1，2和4周龄小鼠发育过程中表达在小胶质细胞中的miR-124相比，成年小鼠（8周龄）时的水平。 （A，B）实验进行一式三份平均±3个独立的PCR反应SD显示。 （三）在实验进行重复表示。

<ol>
	<li>Tremblay, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Microglia+in+the+Healthy+Brain.">The Role of Microglia in the Healthy Brain.</a> J. Neurosci. 31, 16064-16069 (2011).</li><li>Graeber, M. B., Li, W., Rodriguez, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microglia+in+CNS+inflammation.">Role of microglia in CNS inflammation.</a> FEBS Lett. , (2011).</li><li>Ponomarev, E. D., Shriver, L. P., Maresz, K., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cell+activation+and+proliferation+precedes+the+onset+of+CNS+autoimmunity.">Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.</a> J. Neurosci. Res. 81, 374-389 (2005).</li><li>Perruisseau-Carrier, C., Jurga, M., Forraz, N., McGuckin, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+stem+cell+reprogramming+for+neuronal+induction+and+differentiation.">miRNAs stem cell reprogramming for neuronal induction and differentiation.</a> Mol. Neurobiol. 43, 215-227 (2011).</li><li>McCoy, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+miRNAs+in+cytokine+signaling.">The role of miRNAs in cytokine signaling.</a> Front Biosci. 17, 2161-2171 (2010).</li><li>He, M., Xu, Z., Ding, T., Kuang, D. M., Zheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-155+regulates+inflammatory+cytokine+production+in+tumor-associated+macrophages+via+targeting+C/EBPbeta.">MicroRNA-155 regulates inflammatory cytokine production in tumor-associated macrophages via targeting C/EBPbeta.</a> Cell Mol. Immunol. 6, 343-352 (2009).</li><li>Ponomarev, E. D., Veremeyko, T., Barteneva, N., Krichevsky, A. M., Weiner, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-124+promotes+microglia+quiescence+and+suppresses+EAE+by+deactivating+macrophages+via+the+C/EBP-alpha-PU.1+pathway.">MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-alpha-PU.1 pathway.</a> Nat. Med. 17, 64-70 (2011).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Specific+Cell+Population+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS).</a> J. Vis. Exp. (41), e1546-3791 (2010).</li><li>Gabriely, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+21+promotes+glioma+invasion+by+targeting+matrix+metalloproteinase+regulators.">MicroRNA 21 promotes glioma invasion by targeting matrix metalloproteinase regulators.</a> Mol. Cell Biol. 28, 5369-5380 (2008).</li><li>Caffarelli, E., Filetici, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+in+cancer+development.">Epigenetic regulation in cancer development.</a> Front Biosci. 17, 2682-2694 (2011).</li><li>Cardona, A. E., Huang, D., Sasse, M. E., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+microglial+cells+for+RNA+analysis+or+flow+cytometry.">Isolation of murine microglial cells for RNA analysis or flow cytometry.</a> Nat. Protoc. 1, 1947-1951 (2006).</li><li>Gordon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+magnetic+separation+method+for+high-yield+isolation+of+pure+primary+microglia.">A simple magnetic separation method for high-yield isolation of pure primary microglia.</a> J. Neurosci. Methods. 194, 287-296 (2011).</li><li>Moltzahn, F., Hunkapiller, N., Mir, A. A., Imbar, T., Blelloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Throughput+MicroRNA+Profiling:+Optimized+Multiplex+qRT-PCR+at+Nanoliter+Scale+on+the+Fluidigm+Dynamic+ArrayTM+IFCs.">High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs.</a> J. Vis. Exp. (54), e2552 (2011).</li></ol>

没有利益冲突的声明。

最近显着的利益，考虑到小胶质细胞，这些细胞参与神经发炎和神经退行性疾病相关的许多病症。此外，在免疫系统和癌细胞分化的microRNA的作用显着增长在过去几年5,10。在非激活或休息的小胶质细胞的表型，在7正常的中枢神经系统的维护，我们曾报道了miR-124的作用，但其他类型的小分子RNA在小胶质细胞的表型和激活状态的作用仍然被发现。这将需要开发专门测量中的小胶质细?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 1×PBS </td><td> GIBCO公司</td><td> 14190 </td><td>可制取干元件实验室，pH值7.2-7.4 </td></tr><tr><td> 10X PBS </td><td> Thermo Scientific的</td><td> SH30258.02 </td><td>可制取干元件实验室，pH值7.2-7.4 </td></tr><tr><td> DMEM培养液，胎牛血清</td><td> GBCO </td><td> 11965，10438 </td><td></td></tr><tr><td> miRVana套件</td><td> Invitrogen公司</td><td> AM1561 </td><td></td></tr><tr><td> Percoll密度</td><td>西格玛</td><td> P4937-500毫升</td><td> 100毫升100％的Percoll准备通过混合10毫升10倍PBS和90毫升的Percoll自Sigma公司。 50毫升70％的Percoll准备35毫升100％的Percoll混合15毫升的DMEM 50毫升40％的Percoll准备通过混合20毫升100％的Percoll，30毫升的1X PBS </td></tr><tr><td> MOG 35-55肽</td><td> AnaSpec公司</td><td> 60130-5 </td><td></td></tr><tr><td>终审法院</td><td> DIFCO </td><td> 263810 </td><td></td></tr><tr><td>百日咳毒素</td><td>西格玛</td><td> P7208 </td><td></td></tr><tr><td> FCR封闭抗体</td><td> BD公司</td><td> 553142 </td><td>克隆2.4G2 </td></tr><tr><td>抗CD11b -体育单克隆抗体</td><td> BD公司</td><td> 553311 </td><td>可以用不同的荧光（如AF488） </td></tr><tr><td>抗CD45-APC-Cy7单克隆抗体</td><td> Biolegend </td><td> 103116 </td><td>可以用不同的荧光（如APC）的</td></tr><tr><td>多聚甲醛甲醛（16％） </td><td>环境管理体系公司</td><td> 15710 </td><td>稀释1×PBS 1:15 </td></tr><tr><td> 15％，简称TBE-尿素凝胶</td><td>生命科技公司</td><td> EC68855BOX </td><td></td></tr><tr><td>的TaqMan microRNA的反转录试剂盒</td><td>生命科技股份有限公司</td><td> 4366596 </td><td></td></tr><tr><td>的TaqMan通用PCR扩增预混</td><td>生命科技股份有限公司</td><td> 4304437 </td><td></td></tr><tr><td>的TaqMan microRNA的检测MMU的miR-124A </td><td>生命科技股份有限公司</td><td> 4427975编号001182 </td><td></td></tr><tr><td>的TaqMan microRNA的检测的snoRNA-55 </td><td>生命科技股份有限公司</td><td> 4427975编号001228 </td><td></td></tr><tr><td>无核酸酶水</td><td>生命科技股份有限公司</td><td> AM9937 </td><td>无核酸酶水</td></tr><tr><td> 384清晰的光学反应板</tD&gt; <td>生命科技公司</td><td> 4309849 </td><td>在不同的实时PCR仪器可以取代其他板块（如96孔板） </td></tr><tr><td> dounce均质（15毫升） </td><td>惠顿科学产品。 </td><td> 358044 </td><td>铁氟龙/玻璃</td></tr><tr><td> 40μ的尼龙细胞过滤</td><td>鹘</td><td> 352340 </td><td></td></tr><tr><td>大离心机</td><td> sorval </td><td>逆转录6000B </td><td>转子直径18.5厘米的</td></tr><tr><td>小离心机</td><td> Eppendorf公司</td><td> 5415ŕ </td><td>转子直径6.5厘米的</td></tr><tr><td>实时PCR仪</td><td>生命科技公司</td><td> AB 7900羟色胺</td><td>与其他仪器可以取代</td></tr><tr><td>流式细胞仪</td><td>屋宇署Biosciencess </td><td> LSR的二</td><td>与其他仪器可以取代</TD&gt; </tr><tr><td>流式细胞仪</td><td> BD公司</td><td> FACSAria </td><td>与其他仪器可以取代</td></tr><tr><td>的NanoDrop分光光度计</td><td> Thermo Scientific的</td><td>的NanoDrop 1000 </td><td></td></tr></tbody></table>

detection of micrornas in microglia by real-time pcr in normal cns and during neuroinflammation

小胶质细胞是驻留在中枢神经系统（CNS）的髓系细胞。这些细胞中发挥了重要作用，在许多疾病的病理与神经发炎，如多发性硬化症（MS）2。在一个正常的中枢神经系统胶质细胞表达巨噬细胞标记CD11b和休息型，如活化标志CD45的表达水平低的表现。在中枢神经系统的病理事件中，成为激活小胶质细胞所确定的上调CD45和其他标记3。在中枢神经系统的小胶质细胞的表型和功能的影响因素没有得到很好的研究。小分子RNA（miRNAs）是一个保守分子（〜22个核苷酸长），在许多正常的生理过程，如细 ​​胞生长和分化的4和5炎症等病症的越来越多的家庭。 miRNA的某些靶基因的表达下调的结合互补序列IR基因和发挥了重要作用，在激活先天免疫细胞，包括巨噬细胞和小胶质细胞7。为了调查定义的小胶质细胞的表型，生物功能，以区别于其他类型的巨噬细胞的小胶质细胞的miRNA介导的途径，它是重要的，特别是在居民中枢神经系统的胶质细胞不同亚群的microRNA表达定量评估。测量在中枢神经系统表达的miRNA的常用方法包括定量PCR技术，从整个神经组织和原位杂交。然而，从整个组织匀浆PCR定量不允许在小胶质细胞的miRNA表达的评估，这代表只有5-15％的神经组织的细胞。 原位杂交允许评估组织切片中的特定类型的细胞的microRNA表达，但这种方法是不完全定量。在这份报告中，我们描述了定量和SENSitive实时PCR检测中的小胶质细胞在正常的中枢神经系统或神经发炎使用实验性自身免疫性脑脊髓炎（EAE）小鼠模型，为MS分离miRNA的方法。所描述的方法将是有益的，在正常的中枢神经系统，或在与各种病症，包括MS，中风，跌打损伤，阿尔茨海默氏症和脑肿瘤相关的神经发炎的小胶质细胞的microRNA表达水平来衡量的。

Watch this Scientific Journal Video about Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation at JoVE.com

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

小胶质细胞的居住提供第一线的防御和免疫监视中枢神经系统的巨噬细胞。 microRNA是调节分子，包括巨噬细胞的激活和分化的许多生理过程中发挥了重要作用。在这篇文章中，我们描述了在小胶质细胞的小分子RNA的测量方法。

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

detection-micrornas-microglia-real-time-pcr-normal-cns-during

Research

JoVE Journal

Immunology and Infection

19.3K 次观看.  Harvard Medical School. 小胶质细胞的居住提供第一线的防御和免疫监视中枢神经系统的巨噬细胞。 microRNA是调节分子，包括巨噬细胞的激活和分化的许多生理过程中发挥了重要作用。在这篇文章中，我们描述了在小胶质细胞的小分子RNA的测量方法。

视频: Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

科研

免疫与感染

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T细胞受体删除环同时定量（TRECs）和K-删除重组删除环（KRECs）通过实时PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

实时颤振诱发的粪便中 CWD 朊的转化检测

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...