Abstract：
Optimization of flow cytometry assays for extracellular vesicles (EVs) often fail to include appropriate reagent titrations - the most critically antibody titration is either not performed or is incomplete. Using nonoptimal antibody concentration is one of the main sources of error leading to a lack of reproducible data. Antibody titration for the analysis of antigens on the surface of EVs is challenging for a variety of technical reasons. Using platelets as surrogates for cells and platelet-derived particles as surrogates for EV populations, we demonstrate our process for antibody titration, highlighting some of the key analysis parameters that may confound and surprise new researchers moving into the field of EV research. Additional care must be exercised to ensure instrument and reagent controls are utilized appropriately. Complete graphical analysis of positive and negative signal intensities, concentration, and separation or stain index data is highly beneficial when paired with visual analysis of the cytometry data. Using analytical flow cytometry procedures optimized for cells for EV analysis can lead to misleading and nonreproducible results.
摘要：
细胞外囊泡(EV)的流式细胞术测定的优化通常不能包括适当的试剂滴定-最关键的抗体滴定要么不进行或不完全。使用非最佳抗体浓度是导致缺乏可重复数据的主要错误来源之一。用于分析EV表面上的抗原的抗体滴定由于各种技术原因而具有挑战性。使用血小板作为细胞的替代品，使用血小板衍生的颗粒作为EV群体的替代品，我们展示了我们的抗体滴定过程，突出了一些关键的分析参数，这些参数可能会让进入电动汽车研究领域的新研究人员感到困惑和惊讶。必须格外小心，以确保适当使用仪器和试剂控制。正负信号强度的完整图形分析，浓度,当与细胞计数数据的视觉分析配对时,分离或染色指数数据是非常有益的。使用针对EV分析的细胞优化的分析流式细胞术程序可能导致误导和不可重复的结果。
