We use an electrochemical platform, nanoparticle tracking analysis, and differential centrifugation of single catecholamine vesicles to study the properties of nanometer transmitter vesicles, including the number of molecules, size, and catecholamine concentration inside. Vesicle impact electrochemical cytometry (VIEC) was used to quantify the catecholamine content of single vesicles in different batches isolated from pheochromocytoma (PC12) cells with different ultracentrifugation speeds. We show that, vesicles containing less catecholamine are obtained at subsequent centrifugation steps with higher speed (force). Important to quantification, the cumulative content after subsequent centrifugation steps is equivalent to that of one-step centrifugation at the highest speed, 70 000g. Moreover, as we count molecules in the vesicles, we compared molecular numbers from VIEC, flow VIEC, and intracellular VIEC to corresponding vesicle size measured by nanoparticle tracking analysis to evaluate catecholamine concentration in vesicles. The data suggest that vesicular catecholamine concentration is relatively constant and independent of the vesicular size, indicating vesicular transmitter content as a main factor regulating the vesicle size.

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从PC12细胞分离的单个纳米级囊泡中变送器浓度的电化学定量

我们使用电化学平台，纳米颗粒跟踪分析和单个儿茶酚胺囊泡的离心分离来研究纳米递质囊泡的性质，包括分子数，大小和儿茶酚胺浓度。囊泡冲击电化学细胞计数法（VIEC）用于量化从不同嗜铬细胞瘤（PC12）细胞以不同超速离心速度分离的不同批次中单个囊泡的儿茶酚胺含量。我们表明，含有较少儿茶酚胺的囊泡是在随后的离心步骤中以较高的速度（力）获得的。对于定量分析很重要，后续离心步骤后的累积含量相当于最高速度下的一步离心法，即70000 g。此外，当我们对囊泡中的分子进行计数时，我们将来自VIEC，流动VIEC和细胞内VIEC的分子数与通过纳米粒子跟踪分析测量的相应囊泡大小进行比较，以评估囊泡中儿茶酚胺的浓度。数据表明，水泡儿茶酚胺浓度相对恒定并且与水泡大小无关，表明水泡递质含量是调节水泡大小的主要因素。