Finkielsztein Ariel, Mascarenhas Lorraine, Butin-Israeli Veronika, Sumagin Ronen
Department of Pathology, Feinberg School of Medicine, Northwestern University.
Department of Pathology, Feinberg School of Medicine, Northwestern University;
J Vis Exp. 2018 Mar 2(133):56949. doi: 10.3791/56949.
Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50-1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.
多形核中性粒细胞衍生的微粒（PMN-MPs）是脂质双层的球形微囊泡，直径范围为50-1000纳米。微粒是细胞间通讯和信号传导机制中一个新出现的重要组成部分。由于其大小和释放性质，直到最近微粒的存在才被忽视。然而，随着技术和分析方法的改进，它们在健康和疾病中的功能现在正在显现出来。这里介绍的方案旨在通过流式细胞术和免疫印迹法分离和表征PMN-MPs。此外，还给出了几个实施示例。这些用于微粒分离的方案快速、低成本，并且不需要使用昂贵的试剂盒。此外，它们允许在分离后对微粒进行标记，以及在微粒释放之前对源细胞进行预标记，使用膜特异性荧光染料进行可视化并通过流式细胞术进行分析。然而，这些方法有几个局限性，包括PMN和微粒的纯度以及对精密分析仪器的需求。需要一台高端流式细胞仪来可靠地分析微粒，并将由于噪声或自发荧光导致的假阳性读数降至最低。所描述的方案可用于分离和定义微粒的生物发生，表征其标志物以及在不同刺激条件下组成的变化。可以利用大小异质性来研究膜颗粒与外泌体的内容物是否不同，以及它们在组织稳态中是否发挥不同的作用。最后，在分离和表征微粒之后，可以探索它们在细胞反应和各种疾病模型（包括与PMN相关的炎症性疾病，如炎症性肠病或急性肺损伤）中的功能。
