Carnell-Morris Pauline, Tannetta Dionne, Siupa Agnieszka, Hole Patrick, Dragovic Rebecca
Malvern Instruments Ltd, Amesbury, UK.
Department of Food and Nutritional Sciences, University of Reading, Reading, RG6 6AP, UK.
Methods Mol Biol. 2017;1660:153-173. doi: 10.1007/978-1-4939-7253-1_13.
Fluorescence nanoparticle tracking analysis (fl-NTA) allows for accurate sizing, counting, and phenotyping of extracellular vesicles (EV). Here, we present two protocols for the analysis of EVs using fl-NTA, highlighting the potential pitfalls and challenges. The first protocol utilizes CellMask Orange™ (CMO) as a general membrane marker to label EVs derived from plasma. The second protocol describes the use of a Qdot-conjugated antibody to identify syncytiotrophoblast (STB)-derived EVs. "Standard" preparations of STB-derived EVs enriched for either microvesicles (STBMV) or exosomes (STBEX), containing a known amount of EV positive for the STB specific antigen placental alkaline phosphatase (PLAP), were also used to optimize fl-NTA camera settings.
荧光纳米颗粒追踪分析（fl-NTA）可对细胞外囊泡（EV）进行精确的大小测定、计数和表型分析。在此，我们展示了两种使用fl-NTA分析EV的方案，突出了潜在的陷阱和挑战。第一种方案利用CellMask Orange™（CMO）作为通用膜标记物来标记源自血浆的EV。第二种方案描述了使用量子点偶联抗体来鉴定源自合体滋养层细胞（STB）的EV。富含微泡（STBMV）或外泌体（STBEX）的STB衍生EV的“标准”制剂，含有已知量的对STB特异性抗原胎盘碱性磷酸酶（PLAP）呈阳性的EV，也被用于优化fl-NTA相机设置。
