Detection of Association and Fusion of Giant Vesicles Using a Fluorescence-Activated Cell Sorter
Detection of Association and Fusion of Giant Vesicles Using a Fluorescence-Activated Cell Sorter
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DOI：
10.1021/la102689v
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发表时间：
2010-10-05
期刊：
影响因子：
3.9
通讯作者：
Yomo, Tetsuya
中科院分区：
文献类型：
--
作者：
Sunami, Takeshi;Caschera, Filippo;Yomo, Tetsuya
我们开发了一种使用荧光激活细胞分类器(FAGS)来评估巨大囊泡融合过程的方法。用三种荧光标记和FAGS技术评价巨泡的结合程度和融合程度。将封装在不同囊泡种群中的两个荧光标记作为关联标记；当这些囊泡关联时，两个独立的标记应在单个检测事件中同时观察到。将被包裹在不同囊泡种群中的猝灭荧光标记和去猝灭剂作为融合标记。当内部水溶液混合时，猝灭的标记物被去猝灭剂释放并发出第三荧光信号。虽然纯POPC囊泡的种群没有表现出可检测到的关联或融合，但在10万个巨型囊泡的种群分析中，与带电的两亲分子相反的种群显示出50%的关联和30%的融合。尽管很大一部分囊泡与少量的带电两亲性分子(摩尔分数为5%，仅与POPC相比)有关，但需要较大数量的带电两亲性分子(25%)来诱导囊泡融合。目前的方法还显示，巨大小泡的联合和融合取决于大小，较大的巨大小泡联合和融合的频率更高。
We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FAGS). Three fluorescent markers and FAGS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100 000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.