ISEVxTECH FCS and PDF data

Lyophilised GFP exosome reference standard provided by ISEVxTech from Merck (Exosome standards, fluorescent recombinant, expressed in human cells | Sigma-Aldrich (sigmaaldrich.com)). <br> 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.

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，该产品为荧光重组型外泌体标准品，由人细胞表达，购自西格玛奥德里奇（Sigma-Aldrich，sigmaaldrich.com）。除非另有说明，所有样品与标准品的数据采集时长均为1分钟；若以水作为稀释剂，则使用高效液相色谱（HPLC）级纯水。将冻干的默克重组细胞外囊泡（rEV）用100µl纯水重悬后置于冰上，后续所有样品处理均严格遵循制造商说明书要求在冰上操作。取每毫升含2.17×10^10个颗粒（P/ml）的质量控制（QC）微球，用纯水按1:100比例稀释至100µl，用于校准纳米分析仪（Nanoanalyzer）并构建用于样品评估的浓度标准。将粒径分别为68nm、91nm、113nm及155nm的二氧化硅纳米球混合标准微球，用纯水按1:100比例稀释至100µl后进行分析，以此构建粒径标准曲线（见图1）。取100µl Tris-EDTA（TE）缓冲液进行分析，作为样品检测的空白对照。使用TE缓冲液将样品依次按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FCS格式保存，相关PDF报告已上传至Figshare平台。