Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms.
Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms.
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DOI：
10.1002/jev2.12079
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发表时间：
2021-04
影响因子：
16
通讯作者：
Witwer KW
中科院分区：
文献类型：
--
作者：
Arab T;Mallick ER;Huang Y;Dong L;Liao Z;Zhao Z;Gololobova O;Smith B;Haughey NJ;Pienta KJ;Slusher BS;Tarwater PM;Tosar JP;Zivkovic AM;Vreeland WN;Paulaitis ME;Witwer KW
我们比较了细胞外囊泡（EVs）和合成颗粒的大小、计数和表型的四种正交技术。平台包括：单粒子干涉反射成像传感（SP‐IRIS）荧光、纳米粒子跟踪分析（NTA）荧光、微流控电阻脉冲传感（MRPS）和纳米流式细胞术测量（NFCM）。采用差示超离心（dUC）或超滤（UF）和大小排斥层析（SEC）相结合的方法，从培养条件培养基（CCM）中分离出人T淋巴细胞系H9（高CD81，低CD63）和祖细胞细胞系U937（低CD81，高CD63）的EVs，并采用透射电镜（TEM）和Western blot （WB）对其进行表征。还测试了已知大小和/或浓度的合成颗粒（二氧化硅和聚苯乙烯球）的混合物。MRPS和NFCM检测到的颗粒数量相似，而NTA检测到的电动汽车颗粒数量大约低一个数量级，但合成颗粒却没有。SP - IRIS事件不能用于估计颗粒浓度。对于尺寸，SP‐IRIS、MRPS和NFCM返回相似的尺寸曲线，较小的尺寸占主导地位（按幂律分布），但灵敏度通常在直径60 nm以下下降。NTA检测到模直径大于100 nm的粒子群。此外，SP‐IRIS、MRPS和NFCM能够在二氧化硅或聚苯乙烯纳米颗粒的混合物中识别出至少四种不同大小的种群。最后，对于四蛋白表型，荧光模式下的SP‐IRIS平台能够在同一颗粒上检测到至少两个标记，而NFCM只能检测到CD81或CD63。基于这项研究的结果，我们可以得出现有的单颗粒分析能力的结论，这可能对EV生物标志物的开发和机制研究有用。
We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single‐particle interferometric reflectance imaging sensing (SP‐IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP‐IRIS events could not be used to estimate particle concentrations. For sizing, SP‐IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP‐IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP‐IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single‐particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.
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影响因子：
16
作者：
Lötvall J;Hill AF;Hochberg F;Buzás EI;Di Vizio D;Gardiner C;Gho YS;Kurochkin IV;Mathivanan S;Quesenberry P;Sahoo S;Tahara H;Wauben MH;Witwer KW;Théry C
通讯作者：
Théry C
DOI：
10.1109/tbme.2013.2272666
发表时间：
2013-12
期刊：
IEEE transactions on bio-medical engineering
影响因子：
--
作者：
Reddington AP;Trueb JT;Freedman DS;Tuysuzoglu A;Daaboul GG;Lopez CA;Karl WC;Connor JH;Fawcett H;Ünlu MS
通讯作者：
Ünlu MS
影响因子：
16
作者：
Bachurski, Daniel;Schuldner, Maximiliane;von Strandmann, Elke Pogge
通讯作者：
von Strandmann, Elke Pogge
影响因子：
3.2
作者：
Osteikoetxea, Xabier;Sodar, Barbara;Buzas, Edit Iren
通讯作者：
Buzas, Edit Iren
影响因子：
16
作者：
Arab, T.;Raffo-Romero, A.;Sautiere, P-E
通讯作者：
Sautiere, P-E