Red de Apoyo a la Investigación, Universidad Nacional Autónoma de México e Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional.
Red de Apoyo a la Investigación, Universidad Nacional Autónoma de México e Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán.
J Vis Exp. 2021 Apr 23(170). doi: 10.3791/61695.
Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method's limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.
细胞外囊泡（EVs）是由任何细胞类型在任何体液（如尿液）中分泌的脂质膜的异质复合物。EVs 的直径可以从 40-100nm（如外泌体）到 100-1000nm（微囊泡）不等，它们还可以包含不同的分子，这些分子可以作为许多疾病的预后和诊断的生物标志物。已经开发了许多技术来表征这些囊泡。其中一种是流式细胞术。然而，目前还没有报道显示如何定量 EVs 的浓度，并按大小区分它们，同时进行生物标志物检测。本工作旨在描述一种使用常规细胞仪进行分析的尿细胞外囊泡（uEVs）的分离、定量和表型分析的程序，而无需对其配置进行任何修改。该方法的局限性包括每个样本最多只能染色四种不同的生物标志物。该方法还受到样本中 EVs 数量的限制。尽管存在这些局限性，但通过本方案及其后续分析，我们可以获得更多关于肾脏和大脑损伤相关疾病中尿液样本中 EVs 标志物富集和这些囊泡丰度的信息。
