Raw data excel and report

Lyophilised GFP exosome reference standard provided by ISEVxTech from Merck (Exosome standards, fluorescent recombinant, expressed in human cells | Sigma-Aldrich (sigmaaldrich.com)). Unless otherwise mentioned all samples and standards had data acquired over one minute and where water is used as a diluent it is HPLC grade water. Lyophilized Merck rEV’s resuspended in 100µl of water then placed on ice (all subsequent sample handling performed on ice as per manufacturer instruction). 2.17E+10 particles per ml (P/ml) QC beads diluted 1 in 100 with water to a volume of 100µl. Used to align Nanoanalyzer and create the concentration standard used to assess the samples. Size bead cocktail (68nm, 91nm, 113nm and 155nm) of silica nanospheres diluted 1 in 100 to a volume of 100µl with water then analysed to create the size standard (figure 1). 100µl TE buffer was analysed in order to provide a blank for the samples. Samples were serially diluted 1 in 25, 1 in 50 and 1 in 100 dilutions with TE buffer then immediately analysed in triplicate. Data analysis performed using the nFCM professional software, files saved in FCS. format with associated PDF reports found on Figshare.

本研究使用由默克（Merck）旗下ISEVxTech提供的冻干绿色荧光蛋白（GFP）外泌体参考标准品，该标准品为荧光重组型、人源表达的外泌体标准品，购自西格玛奥德里奇（Sigma-Aldrich, sigmaaldrich.com）。除非另有说明，所有样本与标准品的数据采集时长均为1分钟；若以水作为稀释剂，则采用高效液相色谱（High Performance Liquid Chromatography, HPLC）级纯水。冻干的默克重组细胞外囊泡（recombinant extracellular vesicles, rEV）经100μl纯水重悬后置于冰上，后续所有样本操作均按照制造商说明书要求在冰上进行。将浓度为2.17×10¹⁰粒子/毫升（P/ml）的质量控制（Quality Control, QC）微球用纯水按1:100比例稀释至总体积100μl，用于校准纳米分析仪，并构建用于评估样本浓度的浓度标准曲线。将粒径分别为68nm、91nm、113nm和155nm的二氧化硅微球混合校准品用纯水按1:100比例稀释至100μl，随后进行分析以构建粒径标准（见图1）。取100μl Tris-EDTA（TE）缓冲液进行分析，作为样本检测的空白对照。将样本用TE缓冲液按1:25、1:50及1:100的比例进行梯度稀释，随后立即进行三次重复检测。数据分析采用nFCM专业软件完成，数据文件以流式细胞术标准（Flow Cytometry Standard, FCS）格式存储，相关PDF报告已上传至Figshare平台。