Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry
Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry
复制标题
DOI：
10.1038/nprot.2012.065
复制
发表时间：
2012-07-01
期刊：
影响因子：
14.8
通讯作者：
Wauben, Marca H. M.
中科院分区：
文献类型：
--
作者：
van der Vlist, Els J.;Nolte-'t Hoen, Esther N. M.;Wauben, Marca H. M.
我们提供了一种基于高分辨率流式细胞术的方法，用于对细胞释放的单个纳米大小的囊泡进行定量和定性分析，正如我们小组之前所开发和描述的那样。该方法包括(I)对细胞来源的小泡进行明亮的荧光标记，以及(Ii)使用商业上可用的BD内流流式细胞仪的优化配置对这些小泡进行流式细胞术分析。该方法允许检测和分析类似于100 nm的荧光细胞来源的囊泡。可以获得关于这些纳米泡的光散射、数量、浮力密度和表面蛋白质的综合信息。这种方法可以应用于纳米生物学，研究细胞衍生囊泡的基本方面。潜在的临床应用包括体液中基于囊泡的生物标志物的详细分析和用作治疗剂的(生物)囊泡的质量控制分析。囊泡的分离、荧光标记和纯化可以在24小时内完成。流式细胞仪的设置、校准和后续数据采集可以在2-4小时内由经验丰富的流式细胞仪操作员完成。
We provide a protocol for a high-resolution flow cytometry-based method for quantitative and qualitative analysis of individual nano-sized vesicles released by cells, as developed and previously described by our group. The method involves (i) bright fluorescent labeling of cell-derived vesicles and (ii) flow cytometric analysis of these vesicles using an optimized configuration of the commercially available BD Influx flow cytometer. The method allows the detection and analysis of fluorescent cell-derived vesicles of similar to 100 nm. Integrated information can be obtained regarding the light scattering, quantity, buoyant density and surface proteins of these nano-sized vesicles. This method can be applied in nanobiology to study basic aspects of cell-derived vesicles. Potential clinical applications include the detailed analysis of vesicle-based biomarkers in body fluids and quality control analysis of (biological) vesicles used as therapeutic agents. Isolation, fluorescent labeling and purification of vesicles can be done within 24 h. Flow cytometer setup, calibration and subsequent data acquisition can be done within 2-4 h by an experienced flow cytometer operator.