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/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 border="0" cellpadding="0" cellspacing="0" width="644">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">unlabeled anti-VSG antibody</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">VSG antibody is made in house</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">fluorophore labeled anti-VSG antibody</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">VSG antibody is made in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HMI-9 media</td>
			<td style="width:143px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td style="width:167px;">HMI-9 is made in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Propidium Iodide</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td>556463</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CountBright absolute counting beads</td>
			<td style="width:143px;">Thermofisher Scientific</td>
			<td>C36950</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LD columns</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS magnet</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS magnetic separator</td>
			<td style="width:143px;">Miltenyi Biotech</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">vortex adapter-60</td>
			<td style="width:143px;">ThermoFisher scientific</td>
			<td>AM10014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">flow cytometer</td>
			<td style="width:143px;">Coulter</td>
			<td style="width:119px;">n/a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">flow cytometer analysis software</td>
			<td style="width:143px;">FloJo</td>
			<td style="width:119px;">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/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/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/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

Nanyang Technological University

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

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

特定细胞群的净化荧光激活细胞分选（FACS）

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

科研

免疫与感染

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

审核端粒G -悬结构锥虫布氏</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

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 ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

荧光激活细胞分选从小鼠骨髓浆树突状细胞的分离纯化

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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