Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?
Measuring Extracellular Vesicles by Conventional Flow Cytometry: Dream or Reality?
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DOI：
10.3390/ijms21176257
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发表时间：
2020-08-29
影响因子：
5.6
通讯作者：
Fattorossi A
中科院分区：
文献类型：
--
作者：
Lucchetti D;Battaglia A;Ricciardi-Tenore C;Colella F;Perelli L;De Maria R;Scambia G;Sgambato A;Fattorossi A
正在使用临床实验室中可用的流式细胞仪进行密集的研究，以评估各种疾病中的细胞外囊泡（EV）表面货物。使用从HT 29人结直肠腺癌细胞系纯化的各种大小的EV，我们报告了通过常规流式细胞仪评估小型和中型EV的困难，该流式细胞仪将405 nm激光的光侧散射与EV通用标记钙黄绿和钙黄绿紫以及表面标记物的荧光信号相结合。小尺寸EV（~70 nm）免疫表型分析失败，与单克隆抗体结合位点的缺乏一致，因此被排除在进一步研究之外。中等大小的EV（~250 nm）免疫表型是可能的，但它们的检测受到过量的重合颗粒（群检测）和高中止率的困扰;这两个因素都会影响测量的EV浓度。通过运行含有等量钙黄绿素和钙黄绿素紫染色的中等大小EV的样品，我们发现群检测产生假双阳性事件，这种现象通过样品稀释显著减少，但不能完全消除。此外，运行高度稀释的样品需要长时间的细胞计数器时间。目前的研究结果提出了常规流式细胞仪EV分析的常规适用性的问题。
Intense research is being conducted using flow cytometers available in clinically oriented laboratories to assess extracellular vesicles (EVs) surface cargo in a variety of diseases. Using EVs of various sizes purified from the HT29 human colorectal adenocarcinoma cell line, we report on the difficulty to assess small and medium sized EVs by conventional flow cytometer that combines light side scatter off a 405 nm laser with the fluorescent signal from the EVs general labels Calcein-green and Calcein-violet, and surface markers. Small sized EVs (~70 nm) immunophenotyping failed, consistent with the scarcity of monoclonal antibody binding sites, and were therefore excluded from further investigation. Medium sized EVs (~250 nm) immunophenotyping was possible but their detection was plagued by an excess of coincident particles (swarm detection) and by a high abort rate; both factors affected the measured EVs concentration. By running samples containing equal amounts of Calcein-green and Calcein-violet stained medium sized EVs, we found that swarm detection produced false double positive events, a phenomenon that was significantly reduced, but not totally eliminated, by sample dilution. Moreover, running highly diluted samples required long periods of cytometer time. Present findings raise questions about the routine applicability of conventional flow cytometers for EV analysis.
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影响因子：
4.6
作者：
Chutipongtanate S;Greis KD
通讯作者：
Greis KD
影响因子：
4.6
作者：
Kibria G;Ramos EK;Lee KE;Bedoyan S;Huang S;Samaeekia R;Athman JJ;Harding CV;Lötvall J;Harris L;Thompson CL;Liu H
通讯作者：
Liu H
影响因子：
10.6
作者：
Lane RE;Korbie D;Hill MM;Trau M
通讯作者：
Trau M
影响因子：
3.7
作者：
Welsh, Joshua A.;Horak, Peter;Englyst, Nicola A.
通讯作者：
Englyst, Nicola A.
影响因子：
5.5
作者：
Welsh JA;Holloway JA;Wilkinson JS;Englyst NA
通讯作者：
Englyst NA