Lukacs-Kornek Veronika, Julich-Haertel Henrike, Urban Sabine Katharina, Kornek Miroslaw
Department of Medicine II, Saarland University Medical Center, Saarland University, Kirrbergerstr. 100, Homburg, 66424, Germany.
Methods Mol Biol. 2017;1660:201-208. doi: 10.1007/978-1-4939-7253-1_16.
Larger extracellular vesicles, microparticles (MPs) or microvesicles (MVs), especially their acquisition and characterization by flow cytometry (FACS), is increasingly in focus of clinical/translational research efforts. Several laboratories have shown that MPs/MVs might be suitable for the diagnosis and predicting prognosis in various diseases including cancer. However, FACS staining of larger extracellular vesicles (EVs) can be difficult and results potentially in false positive and inconsistent data interpretation. Despite that FACS equipment is well maintained and the operators have ample experience, a reliable and for larger EVs optimized staining protocol is missing. Here, we aim to close that gap and provide a working multi-antibody staining protocol for larger EVs isolated from human serum samples. We describe in detail the needed steps as currently done in our laboratory. Staining is demonstrated exemplarily for multi-antibody mix including CD147 , a potential cancer marker if applied in combination with other MP/MV surface markers.
较大的细胞外囊泡，即微粒（MPs）或微泡（MVs），尤其是通过流式细胞术（FACS）对其进行获取和表征，越来越成为临床/转化研究工作的重点。多个实验室已表明，MPs/MVs可能适用于包括癌症在内的各种疾病的诊断和预后预测。然而，对较大的细胞外囊泡（EVs）进行FACS染色可能具有挑战性，并且可能导致假阳性和数据解释不一致。尽管FACS设备维护良好且操作人员经验丰富，但仍缺少一种可靠的、针对较大EVs优化的染色方案。在此，我们旨在填补这一空白，并提供一种适用于从人血清样本中分离出的较大EVs的多抗体染色实用方案。我们详细描述了目前我们实验室所采用的必要步骤。以包含CD147的多抗体混合物为例展示染色过程，CD147如果与其他MP/MV表面标志物联合应用，是一种潜在的癌症标志物。
