BACKGROUND A major concern for the extracellular vesicle (EV) field is the current lack of accurate methods for EV quantification. Total protein measurement fails to reliably quantify EVs from serum-containing conditioned media and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate between membrane-bounded EVs, lipids and protein aggregates. However, EVs can be fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. Fluorescence-based NTA (F-NTA) is thus emerging as a method for counting and phenotyping of EVs. We have validated a differential NTA/F-NTA method using specific antibodies against surface markers in analogy to flow cytometric analyses. METHODS EVs from umbilical cord mesenchymal stromal cells (UC-MSCs) were isolated by a combined tangential flow filtration and ultracentrifugation protocol. EV preparations from 2 x 107 cells were stained with AlexaFluor 488-conjugated specific antibodies or corresponding isotype controls. Amount and size of particles in normal scattering light mode (N mode) versus fluorescence mode (F mode, laser wavelength 488 nm) was measured using ZetaView Nanoparticle Tracking Analyzer (Particle Metrix). Cryo electron microscopy (EM) was used to verify the presence of membrane bilayer surrounded nanoparticles. RESULTS All UC-MSC-EV preparations were found positive for typical EV marker proteins and negative for MHC class I. Novel and improved devices that include more sensitive cameras for detection in the fluorescent mode further increase the detection limit. CONCLUSION Differential NTA/F-NTA facilitates determination of the percentage of EV marker protein-positive nanoparticles within a mixed particulate solution. The set of markers can be extended to other MSC-EV positive and negative surface proteins in order to establish F-NTA-based profiling as a supporting method for the quantification of EVs.

中文翻译：

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用于计算混合颗粒溶液中细胞外囊泡含量的差分荧光纳米颗粒跟踪分析

背景细胞外囊泡 (EV) 领域的一个主要问题是目前缺乏准确的 EV 定量方法。总蛋白测量无法可靠地量化来自含血清条件培养基的 EV，经典纳米粒子跟踪分析 (NTA) 允许对粒子进行量化和大小测定，但无法区分膜结合的 EV、脂质和蛋白质聚集体。然而，EV 可以用非特异性膜标记或特异性识别 EV 表面标记蛋白的抗体进行荧光标记。因此，基于荧光的 NTA (F-NTA) 正在成为一种计算和表型 EV 的方法。我们已经验证了一种差分 NTA/F-NTA 方法，该方法使用针对表面标记的特异性抗体，类似于流式细胞术分析。方法 来自脐带间充质基质细胞 (UC-MSC) 的 EV 通过切向流过滤和超速离心相结合的方案进行分离。来自 2 x 107 个细胞的 EV 制剂用 AlexaFluor 488 偶联的特异性抗体或相应的同种型对照染色。使用 ZetaView Nanoparticle Tracking Analyzer (Particle Metrix) 测量正常散射光模式 (N 模式) 与荧光模式 (F 模式，激光波长 488 nm) 下粒子的数量和大小。冷冻电子显微镜 (EM) 用于验证膜双层包围纳米粒子的存在。结果发现所有 UC-MSC-EV 制剂对典型 EV 标记蛋白呈阳性，对 MHC I 类呈阴性。包括在荧光模式下进行检测的更灵敏相机在内的新型改进设备进一步提高了检测限。结论 差分 NTA/F-NTA 有助于确定混合颗粒溶液中 EV 标记蛋白阳性纳米颗粒的百分比。这组标记可以扩展到其他 MSC-EV 阳性和阴性表面蛋白，以建立基于 F-NTA 的分析，作为量化 EV 的支持方法。