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首页> 外文期刊>Genes to cells : >Flow cytometric quantitation of EpCAM-positive extracellular vesicles by immunomagnetic separation and phospholipid staining method
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Flow cytometric quantitation of EpCAM-positive extracellular vesicles by immunomagnetic separation and phospholipid staining method

机译：免疫磁性分离和磷脂染色法流动细胞计数定量。

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Extracellular vesicles (EV) have attracted attention as circulating biomarkers for many diseases, particularly cancer. Conventional immunofluorescence staining has been used for the detection of target antigens on EV by flow cytometry. However, the staining intensity depends on the amount of antigen expressed on the vesicles and is often only around the noise level. Instead of immunofluorescence, we combined immunomagnetic separation using nanosize MACS (R) MicroBeads with phospholipid staining of EV (IMS-PS method). EpCAM-positive EV were prepared from the culture supernatants of OVCAR3 (EpCAM-high), A431 (EpCAM-low) or Colon-26 (non-human control) cells as cancer models and were examined by the IMS-PS method using EpCAM mAb-coated MicroBeads. By employing Polaric-500c6F as the dye for staining EV phospholipids and using appropriate flow cytometry settings, autofluorescence was excluded, whereas pretreatment of the MicroBeads with conventional blocking agents reduced nonspecific binding to non-target vesicles. These modifications resulted in a linear relation between the number of EV detected and the sample volume, regardless of the level of EpCAM expression on the vesicles. A431 EV spiked into healthy volunteer plasma were enumerated with good accuracy. The IMS-PS method may be useful for clinical evaluation of EV with low levels of antigen expression that are difficult to detect by conventional immunofluorescence.
机译：细胞外囊泡（EV）引起了循环生物标志物的关注，适用于许多疾病，特别是癌症。常规免疫荧光染色已被用于通过流式细胞术检测EV上的靶抗原。然而，染色强度取决于囊泡上表达的抗原的量，并且通常仅在噪声水平周围。除了具有IV的磷脂染色的纳米型MAC（R）微珠（IMS-PS方法），我们将免疫磁分离组合免疫磁性分离。从OVCAR3（EPCAM-HIGH），A431（EPCAM-LOW）或结肠-26（非人类对照）细胞作为癌症模型的培养基阳性EV制备EV，并通过EPCAM MAB的IMS-PS方法检查涂层微珠。通过使用极性-500C6F作为染料染色EV磷脂并使用适当的流式细胞术设置，排除了自发荧光，而常规阻断剂的微珠预处理是与非靶囊泡的非特异性结合。这些修改导致检测到的EV的数量和样品体积之间的线性关系，而不管囊泡上的EPCAM表达水平如何。 A431 EV飙升到健康的志愿等离子体中以良好的准确度列举。 IMS-PS方法可用于EV的临床评估，其具有低水平的抗原表达，难以通过常规免疫荧光检测。

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