检测<em>布氏锥虫</em>变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关
Summary
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.
Abstract
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.
Introduction
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'开关'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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| unlabeled anti-VSG antibody | n/a | n/a | VSG antibody is made in house |
| fluorophore labeled anti-VSG antibody | n/a | n/a | VSG antibody is made in house |
| HMI-9 media | n/a | n/a | HMI-9 is made in house |
| Propidium Iodide | BD Pharmingen | 556463 | |
| CountBright absolute counting beads | Thermofisher Scientific | C36950 | |
| LD columns | Miltenyi Biotech | 130-042-901 | |
| MACS magnet | Miltenyi Biotech | 130-042-303 | |
| MACS magnetic separator | Miltenyi Biotech | 130-042-302 | |
| vortex adapter-60 | ThermoFisher scientific | AM10014 | |
| flow cytometer | Coulter | n/a | |
| flow cytometer analysis software | FloJo | n/a |
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