Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.
Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies.
复制标题
DOI：
10.1038/s41598-017-15717-7
复制
发表时间：
2017-11-10
影响因子：
4.6
通讯作者：
Stassen FRM
中科院分区：
文献类型：
--
作者：
Benedikter BJ;Bouwman FG;Vajen T;Heinzmann ACA;Grauls G;Mariman EC;Wouters EFM;Savelkoul PH;Lopez-Iglesias C;Koenen RR;Rohde GGU;Stassen FRM
适当的分离方法对于阐明细胞外囊泡（EV）和无EV分泌组对稳态和疾病的相对贡献至关重要。我们假设，超滤后的尺寸排阻色谱法（UF-SEC）提供了良好匹配的浓缩物的EV和游离分泌的分子的蛋白质组学和功能研究。将BEAS-2B支气管上皮细胞的条件培养基在10 kDa离心过滤器上浓缩，然后使用琼脂糖CL-4 B SEC分离EV和游离蛋白。或者，通过超离心分离EV。EV恢复估计珠耦合流式细胞术和可调电阻脉冲传感。EV分离物和SEC蛋白级分的蛋白质组组成通过纳米LC-MS/MS表征。UF-SEC EV倾向于具有比超离心EV更高的产率和EV-蛋白质纯度比。UF-SEC EV和超离心EV显示出与UF-SEC蛋白不同的生物学途径的相似富集倍数。用UF-SEC蛋白处理BEAS-2B细胞，而不是用任一类型的EV分离物处理，增加了培养基中的IL-8浓度，而EV，而不是蛋白诱导单核细胞粘附到内皮细胞。因此，UF-SEC是用于超离心的有用替代方案，并且允许比较EV和游离分泌分子的蛋白质组组成和功能效应。
Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules.
登录
查看更多内容
影响因子：
24.3
作者：
Admyre, C;Grunewald, J;Gabrielsson, S
通讯作者：
Gabrielsson, S
DOI：
10.1016/j.nano.2015.01.003
发表时间：
2015-05-01
影响因子：
5.4
作者：
Nordin, Joel Z.;Lee, Yi;Andaloussi, Samir E. L.
通讯作者：
Andaloussi, Samir E. L.
影响因子：
16
作者：
Yáñez-Mó M;Siljander PR;Andreu Z;Zavec AB;Borràs FE;Buzas EI;Buzas K;Casal E;Cappello F;Carvalho J;Colás E;Cordeiro-da Silva A;Fais S;Falcon-Perez JM;Ghobrial IM;Giebel B;Gimona M;Graner M;Gursel I;Gursel M;Heegaard NH;Hendrix A;Kierulf P;Kokubun K;Kosanovic M;Kralj-Iglic V;Krämer-Albers EM;Laitinen S;Lässer C;Lener T;Ligeti E;Linē A;Lipps G;Llorente A;Lötvall J;Manček-Keber M;Marcilla A;Mittelbrunn M;Nazarenko I;Nolte-'t Hoen EN;Nyman TA;O'Driscoll L;Olivan M;Oliveira C;Pállinger É;Del Portillo HA;Reventós J;Rigau M;Rohde E;Sammar M;Sánchez-Madrid F;Santarém N;Schallmoser K;Ostenfeld MS;Stoorvogel W;Stukelj R;Van der Grein SG;Vasconcelos MH;Wauben MH;De Wever O
通讯作者：
De Wever O
影响因子：
16
作者：
Kim DK;Kang B;Kim OY;Choi DS;Lee J;Kim SR;Go G;Yoon YJ;Kim JH;Jang SC;Park KS;Choi EJ;Kim KP;Desiderio DM;Kim YK;Lötvall J;Hwang D;Gho YS
通讯作者：
Gho YS
影响因子：
4.6
作者：
Vergauwen G;Dhondt B;Van Deun J;De Smedt E;Berx G;Timmerman E;Gevaert K;Miinalainen I;Cocquyt V;Braems G;Van den Broecke R;Denys H;De Wever O;Hendrix A
通讯作者：
Hendrix A