我们要感谢乔治十字对锥虫生物学的一般建议。这项工作也由比尔和梅琳达·盖茨基金会GCE授予DS，一个国家科学基金会研究生研究奖学金（DGE-1325261），以MRM和NIH / NIAID（批＃AI085973），以支持FNP。我们感谢凯兰崔尔伽格尔营地，矿工使用含I-SCEI基因识别位点的应变。

注意:在整个过程中，有必要保持细胞在冰上。冷媒体也应在整个使用。 1.样本收获<ol><li>利斯特增长427株血液锥虫至0.5的密度 - 100万/ ml。这是最好的开始与文化少数寄生虫。降速50×10 6细胞/样品在1500×g下10分钟。一定要留下1×10 6个细胞培养后用作阳性和阴性对照抗体。 注:此协议也可以从动物血液（详见讨论）隔离锥虫使用。 </li><li>吸管或倒掉大部分的上清液（留下约750微升）。转移细胞到1.5ml微量离心管中。 </li><li>旋转细胞在4℃下4分钟为5，200 XG在微量和上清。 </li></ol> 2.磁标记<ol><li>悬浮细胞在150微升培养基（HMI-9的Wi例如第血清）+基本抗VSG抗体在适当的稀释度。 注:抗VSG抗体是在内部提出，并在1:50的稀释使用。以使用不带有荧光团标记的初级抗VSG抗体是很重要的。 </li><li>在冷室中在4℃下进行10分钟，8 - 使用涡旋适配器60以速度6涡流细胞。 1000微升冷HMI-9与血清 - 800清洗。 </li><li>旋在在5200 xg离心4℃4分钟，吸出上清液。洗用1ml的HMI-9与血清并如上所述旋转。吸出上清液。重悬沉淀在100μl的HMI-9的血清。 </li><li>添加磁性活化细胞的110微升分选（MSC）的微珠。使用抗小鼠，抗兔或抗生物素珠粒作为适合主抗VSG抗体。拌匀。涡旋细胞在冷室中在4℃下进行10分钟。 </li><li>当细胞被涡旋，建立一个磁性激活分离柱/样本上分离磁铁。一定要有一个重新ceptacle（我们使用一个15毫升的离心管）的列下收集流过。在冷室中设置的装置。 </li><li>加2ml HMI-9与血清素到列。列下吸代替15ml离心管，收集流过每个样品之​​后。 </li><li> 10分钟孵化步骤2.4后，加入800-1000微升冷HMI-9血清。旋在4℃4分钟为5，200 XG和上清以除去未结合的抗体。用1ml的HMI-9与血清和重复留下100μl的上清液洗。 </li><li>轻弹离心管大力和目视检查管，以确保沉淀完全重悬。加入1ml的HMI-9与血清和移液器上下，以确保细胞是公重悬。 </li></ol> 3.磁选<ol><li>确保与上述列灌注。应用细胞列。与细胞中表达的非主导VS收集流通G. 1ml培养基的+锥虫通常需要6 - 7分钟通过该柱流过。 </li><li>洗涤2次用1毫升的HMI-9与血清，并收集在同一个管流过如在步骤3.1。 </li><li>分流通（3毫升）中分成两个1.5ml微量管中。 注意:这些可购买组合或半可用于分离RNA 等 </li><li>从磁铁删除列。与血清添加3毫升HMI-9的柱和沉浸柱入15ml离心管，以获得表达的起始，主导VSG细胞。除去300μl的洗脱材料的供以后分析。置于冰上这些细胞。 </li><li>用作阳性和阴性对照，采取从对照细胞的起始培养大约为1百万个细胞，在37℃生长（那些预计不频繁切换）并吸取它们放入一个1.5ml微量管中。 注意:阳性对照细胞将与荧光标记的抗体在步骤4.1染色。阴性对照组REMA在整个协议的其余部分未染色。 </li><li>旋流过的样品和在5200 xg离心在4℃下4分钟阳性抗体对照样品并除去上清液。 注:洗脱细胞的另一300μl的等分试样可被用作用于抗VSG染色的阳性对照。 </li></ol> 4.染色识别污染物和切换器<ol><li>悬浮流通的样品和在100微升的HMI-9与血清+伯，荧光团标记，抗VSG在适当的稀释抗体的阳性对照细胞。 注意:标记的抗VSG抗体是在内部制成，以1:1的稀释比使用:1000。所述荧光团标记，抗VSG可从同一源如在MACS分离未标记的抗体step.There应在以下步骤3.5 2离心管3毫升流过的样品。 </li><li>分析所有的流通过流式细胞术，结合粒料在两个离心管的同时重新SUS待决100微升HMI-9与血清+抗VSG抗体样品。拌匀。 </li><li>涡旋细胞在冷室中在4℃下进行10分钟。添加800 - 1000微升冷HMI-9血清。在4℃下4分钟旋转样品在5200 xg离心并除去上清液以除去未结合的抗体。 </li><li>洗涤细胞用1ml的HMI-9的血清。在4℃下4分钟旋转样品为5，200 XG和上清。在这个最后的旋转，从步骤3.4和阴性对照样品降速洗脱样本。 </li><li>重悬所有流通和洗脱的样品中:148.5微升HMI-9与血清，25微升绝对算珠和1.5微升碘化丙啶染色溶液（175微升总）。悬浮阳性和阴性抗体对照样本中175微升HMI-9血清。一定要记录绝对算珠/ 25微升所使用的特定批次的数量。 </li><li>贯穿流的所有样本仪和收集数据。作为染色PI的死细胞<sup&gt; +。污染物污染的PI - VSG +。切换器作为染色PI - VSG - 8。 </li><li>计算开关电源采用流式细胞仪数据的频率。 注:有关如何计算的开关频率的说明，请参阅补充文件<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54715/Flow_Cytometry_Calculations.docx" target="_blank">流式细胞仪Calculations.docx</a>和表1。 </li></ol>

<ol>
	<li>Horn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+variation+in+African+trypanosomes.">Antigenic variation in African trypanosomes.</a> Molecular &amp; Biochemical Parasitology. 195 (2), 123-129 (2014).</li><li>Lamont, G. S., Tucker, R. S., Cross, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+antigen+switching+rates+in+Trypanosoma+brucei.">Analysis of antigen switching rates in Trypanosoma brucei.</a> Parasitology. 92 (Pt 2), 355-367 (1986).</li><li>Rudenko, G., McCulloch, R., Dirks-Mulder, A., Borst, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+exchange+can+be+an+important+mechanism+of+variant+surface+glycoprotein+gene+switching+in+Trypanosoma+brucei.">Telomere exchange can be an important mechanism of variant surface glycoprotein gene switching in Trypanosoma brucei.</a> Mol Biochem Parasitol. 80 (1), 65-75 (1996).</li><li>Turner, C. M., Barry, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+of+antigenic+variation+in+Trypanosoma+brucei+rhodesiense+infections.">High frequency of antigenic variation in Trypanosoma brucei rhodesiense infections.</a> Parasitology. 99 (Pt 1), 67-75 (1989).</li><li>Miller, E. N., Turner, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+antigenic+types+appearing+in+first+relapse+populations+of+clones+of+Trypanosoma+brucei.">Analysis of antigenic types appearing in first relapse populations of clones of Trypanosoma brucei.</a> Parasitology. 82 (1), 63-80 (1981).</li><li>Horn, D., Cross, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Trypanosoma+brucei+vsg+expression+site+switching+in+vitro.">Analysis of Trypanosoma brucei vsg expression site switching in vitro.</a> Mol Biochem Parasitol. 84 (2), 189-201 (1997).</li><li>Figueiredo, L. M., Janzen, C. J., Cross, G. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+histone+methyltransferase+modulates+antigenic+variation+in+African+trypanosomes.">A histone methyltransferase modulates antigenic variation in African trypanosomes.</a> PLoS Biol. 6 (7), e161 (2008).</li><li>Boothroyd, C. E., Dreesen, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast-endonuclease-generated+DNA+break+induces+antigenic+switching+in+Trypanosoma+brucei.">A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei.</a> Nature. 459 (7244), 278-281 (2009).</li><li>Engstler, M., Pfohl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+Flow-Mediated+Protein+Sorting+on+the+Cell+Surface+of+Trypanosomes.">Hydrodynamic Flow-Mediated Protein Sorting on the Cell Surface of Trypanosomes.</a> Cell. 131 (3), 505-515 (2007).</li><li>Mugnier, M. R., Cross, G. A. M., Papavasiliou, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+dynamics+of+antigenic+variation+in+Trypanosoma+brucei.">The in vivo dynamics of antigenic variation in Trypanosoma brucei.</a> Science. 347 (6229), 1470-1473 (2015).</li></ol>

The authors have nothing to disclose.

相对于实验技术，该协议的最关键的部件是保持所有样品冷。锥虫非常迅速内在化结合到其表面9抗体，但该方法是运动相关的，只要将细胞保持在4℃下不影响测定。所有样品应始终保持在冰上，并移液应该迅速完成，以减少暴露于25℃的实验室环境中。冷的HMI-9与血清应可在实验的开始，并应在整个保持在冰上。通过在柱运行它们的锥虫的隔离必须在冷室中进行的，因为它需要5 - 7分钟通过该柱以运行一个1ml样品。如果有比可用磁分离单元更多的样品，磁隔离可以以序列为不被积极分离样品保持在冰上进行，如长。 这也是至关重要的样品是非常充分混合自动对焦之三在与微珠和前隔离的磁铁孵化最后一次洗涤。这可通过将离心管内或光涡旋的侵略性轻弹来完成。细胞的无丸粒或团块应加入样品到分离柱，其可通过观察光镜水平进行检查之前是可见的。如果团块存在时，污染的细胞的流过样品中表达显性VSG的水平通常比期望的要高得多。当在协议结束时的样品中切换的数量进行计算，它是否被用于流式细胞仪分析所有的流过，考虑到是很重要的，或者是否使用一半。如果只使用一半，计算切换器的总数应乘以2。 当在洗涤步骤除去上清液，这是没有必要对移液器关闭每一滴，并且通常为15 - 25微升是左周围细胞的沉淀。这是在染色和洗涤的最后阶段特别关键，因为在这一点上粒料流过样品中很少见到，因为有这么几个切换细胞。 而最终反VSG污渍通常与纯化的，荧光团标记的抗VSG抗体进行之前，磁隔离的第一抗VSG染色可与纯化的抗体或血清进行。我们也使用来自表达抗VSG抗体的杂交瘤衍生的生物反应器的上清液。无论如何，用于初始污点的材料必须被滴定以确保或不表达的主导，起始VSG细胞良好的分离。 它是典型的在流通细胞群与表达显性VSG细胞污染。尽管这种污染应该是最小的，在我们的经验，这是罕见的人口是完全没有细胞的表达ING占主导地位的VSG。同样重要的是要注意，已经通过分离过程去细胞不与抗VSG抗体作为那些没有通过程序被作为明亮染色。我们假设两件事情可以解释这种差异。首先，纺丝锥虫导致VSG的脱落，而且还有所涉及的分离步骤在许多旋转。这预期会降低信号的强度的最终反VSG污点。第二个是，该协议的第一步骤涉及染色与伯抗VSG抗体。如果在该第一步骤中使用的抗体识别的表位是相同或阻塞通过在最终的染色的抗体识别的表位，人们会期望的信号的强度为比如果只有一个抗体被用于低。由于这个原因，以滴定是在最后步骤中使用的抗体，例如，从一个染色阳性细胞的信号是大致二是重要幅度比一个负染细胞更高的数量级。这样一来，即使信号从一个染色阳性细胞的强度在已经经历了分离过程的细胞被减小，这些阳性细胞将仍然很好从在流式细胞情节的阴性细胞分离。如果正在使用自动补偿，单染色的对照样品，应制备。我们已经生成的抗体可从纪念斯隆癌症中心的单克隆抗体设施购买。我们曾使用未标记的单克隆IgG抗体的最佳成功。我们已经进行了该协议使用多克隆VSG抗体，但多克隆抗体可以偶尔认识到其它的VSG所以单克隆抗体是优选的。 VSG同一性可以通过从VSG 3&#39;UTR和剪接前导保守序列的扩增产生测序VSG的cDNA来确定。特定抗体可以特异性被测试的先前孤立切换已如刚才所述确定细胞谁的VSG身份。 这里介绍的协议具有许多优点。它可以用来既以分离供以后分析切换器和量化给定群体切换的数量。它也可以用于分离感兴趣的特定变体，只要抗体是可用于该变体。另外，也可以进行使用从动物中分离锥虫这个协议中，前提是红血细胞根据制造商的方案使用抗TER119涂覆磁性珠粒消除。我们没有测试所需锥虫数量的下限，但我们已经成功地进行了使用2.5协议年 - 1000万元锥虫从250微升血液。最后，该协议可以结合使用与波动分析以获得在给定种群的开关频率。 分离过程是相当˚FAST，服用3 - 4小时，从开始到取决于样品的数量完成，从而比以前的所需含药物抗性标记鼠或专门菌株的现有免疫方法更有效。该方法是由针对起始VSG抗体然而需要，这一事实的限制。如果没有这样的试剂，诸如VSG-SEQ的另一种方法可能是更合适的选择，以了解它们在一个给定的群体10被表达的VSG。

原生动物寄生虫布氏锥虫 ，是影响人类（经冈比亚布氏锥虫和布氏锥虫罗得西亚），在整个撒哈拉以南非洲地区和动物（通过布氏锥虫布氏 ）疾病的病原体。它是通过采采蝇矢量的唾液传递到哺乳动物宿主。人类和动物非洲锥虫引起流行地区严重的经济负担，而一些药物可或开发治疗疾病的两种。了解免疫逃逸机制是药物开发锥虫病的关键。茂密，变体表面糖蛋白（VSG）大衣覆盖寄生虫的抗原变异是主要手段之一，由T.布氏逃避哺乳动物抗体反应。将VSG基因的约2000个变体在锥虫基因组中存在，但只有一个在任何给定时间从一个转录〜15 EXPRES锡永的网站。所表达的VSG的&#39;开关&#39;可以通过复制VSG基因进入活性表达位点，或通过先前无声表达位点（1综述）的转录激活发生。 虽然大量的工作一直致力于理解抗原变异的T.布氏 ，影响开关频率的因素仍然知之甚少。迄今为止的研究受到阻碍的事实，而开关在体外随机发生，开关频率非常低，为1在约10 6个细胞2的量级。这使得难以测量给定因子增加或是否减小开关频率，因为切换是硬首先被检测到。在2009年之前，用于在给定的群体中分离切换方法是冗长且劳动密集的。这些措施包括通过传代对主要的VSG免疫小鼠锥虫，然后收获细胞一天后3，或者通过免疫4,5计数数百细胞。另一种策略依赖于选择耐药隔离切换6。因为在体外生长非洲锥虫通常由大量人口表达一个主要变型中，并表达交替变体切换的小得多的人口，我们指的是主要变体在整个本文作为主导，开始VSG。这样做，我们绝不想暗示这主要变种比在其他人群更大的变体健身。 在这里，我们描述了可以可靠地测量锥虫的在3在给定的群体中表达非主导VSG数量的方法 - 4小时。此方法是当一个人想要确定一个给定的基因操作或药物治疗是否增加群交换的细胞的数目为特别有用。相反，摆脱细胞群体表达的做minant，起动VSG通过药物或通过免疫学手段，这些细胞通过首先与耦合到抗体对占优势的VSG磁珠涂覆它们，然后在磁性柱中分离它们消除。开关人口然后在流过收集，并用荧光团标记的抗VSG抗体识别的污染物再次染色。量化是通过将在规定的绝对计数珠号每个样品，使珠细胞的比率可确定并用于量化在人口7切换的数目来实现的。

<table><tbody><tr><td>unlabeled anti-VSG antibody</td><td>n/a</td><td>n/a</td><td>VSG antibody is made in house</td></tr><tr><td>fluorophore labeled anti-VSG antibody</td><td>n/a</td><td>n/a</td><td>VSG antibody is made in house</td></tr><tr><td>HMI-9 media</td><td>n/a</td><td>n/a</td><td>HMI-9 is made in house</td></tr><tr><td>Propidium Iodide</td><td>BD Pharmingen</td><td>556463</td><td></td></tr><tr><td>CountBright absolute counting beads</td><td>Thermofisher Scientific</td><td>C36950</td><td></td></tr><tr><td>LD columns</td><td>Miltenyi Biotech</td><td>130-042-901</td><td></td></tr><tr><td>MACS magnet</td><td>Miltenyi Biotech</td><td>130-042-303</td><td></td></tr><tr><td>MACS magnetic separator</td><td>Miltenyi Biotech</td><td>130-042-302</td><td></td></tr><tr><td>vortex adapter-60</td><td>ThermoFisher scientific</td><td>AM10014</td><td></td></tr><tr><td>flow cytometer</td><td>Coulter</td><td>n/a</td><td></td></tr><tr><td>flow cytometer analysis software</td><td>FloJo</td><td>n/a</td><td></td></tr></tbody></table>

detection of trypanosoma brucei variant surface glycoprotein switching by magnetic activated cell sorting and flow cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

使用此处所描述的方法以证明VSG表达位点内的双链断裂的人口8增加切换的数量。在这里，我们显示已同样诱导产生在表达部位的双链断裂锥虫的群体代表性的结果。我们比较这些锥虫那些没有被诱导以产生双链断裂。 图1显示了代表性的流式细胞仪从流通从磁激活细胞分选柱收集的未诱导和诱导的细胞图。对于这个特定的实验中的主要起始VSG是VSG2。 图1A的左侧面板显示前向和侧向散射，它允许门绕绝对计数珠和细胞，用于显示正向和侧向散射签名是活细胞的特征性绘制。需要注意的是门中被吸引到包括更多的细胞，因为死细胞可以与PI染色被淘汰;然而，栅极必须保持对所有样品是相同的。 图1A的右侧面板只显示落在左侧面板中的"活"的大门内的细胞。该染色积极为PI（Q1和Q2）细胞是死细胞。即积极染色为主导VSG和消极的PI在三季度细胞污染物。这两个PI和Q4的主导VSG负染色细胞是已转活细胞。人们可以看到，在第三季度污染物的百分比是那些双链断裂已诱导细胞低。然而，在第三季度百分比不能用来推断是否有一个给定的群体更切换器。这只是在Q4 的细胞数的比率进行比较，以收集一个可以计算的开关频率的小珠的数量如表1所示从FLO得到的数字W¯¯流式细胞仪的情节和方法来计算这些人群的百分比切换。这些计算是在3个生物样品进行，以获得在图1B中显示的观察开关频率。 在体外随机切换的水平相当低，如这里在平均9.53×10 -5计算，而在1诱导断裂的结果-已切换到非主导VSG 6×10 -3的细胞。 <img alt="图1" src="/files/ftp_upload/54715/54715fig1.jpg" /> 图1. 隔离和交换锥虫种群量化。 （A）流式细胞表示在下面的非切换的上一个磁激活细胞分选列选择流通级分锥虫的级分。与诱导的I-SCEI双链断裂细胞群相比较的细胞群没有诱导冰川AK。（B）的计算值的开关频率对于具有或没有被诱导与I-SCEI双链断裂细胞。在图上的数字代表一个双尾，非配对t检验的结果。误差线代表SD。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54715/54715fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 补充文件1.流式细胞仪计算。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54715/Flow_Cytometry_Calculations.docx" target="_blank">请点击此处下载此文件。</a> <img alt="表格1" src="/files/ftp_upload/54715/54715tbl1.jpg" /> 表1.开关频率的计算。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54715/54715tbl1large.jpg" target="_blank">请点击这里查看此表的放大版本。</a>

Watch this Scientific Journal Video about Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry at JoVE.com

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

detection-trypanosoma-brucei-variant-surface-glycoprotein-switching

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

8.7K 次观看.  Rockefeller University. African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

视频: Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens7. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to na&iuml;ve counterparts7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.
Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to na&iuml;ve MDSC 13. MDSC percentages are significantly less in lymphoid compartments of na&iuml;ve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1+ leukocytes from na&iuml;ve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1+ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1+ leukocytes from spleens of na&iuml;ve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas na&iuml;ve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from na&iuml;ve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1+ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1+ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (na&iuml;ve vs. tumor- bearing) in in vivo and in vitro assays.

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

This is a rapid and comprehensive method of immunophenotyping Myeloid Derived Suppressor Cells (MDSC) and enriching Gr-1+ leukocytes from mouse spleens. This method uses flow cytometry and AutoMACS Cell Sorting to enrich for viable Gr-1+ leukocytes prior to FACS sorting of MDSC for use in vivo and in vitro assays.

制备髓源性抑制细胞从幼稚和胰腺癌荷瘤小鼠，用流式细胞仪和自动磁性细胞分选（AutoMACS）（MDSC）

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

科研

医学

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

免疫与感染

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

在啮齿动物疟原虫原虫的体内评估和裂殖子入侵流式细胞仪

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

生物学

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size 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 ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

阴离子交换剂 (二乙基氨基乙基纤维素柱) 纯化包括非洲在内的细胞外锥虫体

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

细针吸入检测血管外胰腺瘤寄生虫

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

检测

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