Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Nat Protoc. 2012 Jun 14;7(7):1311-26. doi: 10.1038/nprot.2012.065.
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 ∼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.
我们提供了一种基于高分辨率流式细胞术的方法，用于定量和定性分析细胞释放的个体纳米大小囊泡，该方法由我们小组开发并先前描述过。该方法包括 (i) 细胞来源囊泡的明亮荧光标记，以及 (ii) 使用市售的 BD Influx 流式细胞仪的优化配置对这些囊泡进行流式细胞术分析。该方法允许检测和分析约 100nm 的荧光细胞来源囊泡。可以获得有关这些纳米大小囊泡的光散射、数量、浮力密度和表面蛋白的综合信息。该方法可应用于纳米生物学，以研究细胞来源囊泡的基本方面。潜在的临床应用包括对体液中基于囊泡的生物标志物进行详细分析，以及对作为治疗剂使用的 (生物) 囊泡进行质量控制分析。囊泡的分离、荧光标记和纯化可以在 24 小时内完成。经验丰富的流式细胞术操作员可以在 2-4 小时内完成流式细胞仪设置、校准和后续数据采集。
