Estimation of protein and metabolite release rates from damaged mammalian cells by using GFP as a marker molecule
Estimation of protein and metabolite release rates from damaged mammalian cells by using GFP as a marker molecule
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使用 GFP 作为标记分子估计受损哺乳动物细胞的蛋白质和代谢物释放率
DOI：
10.1007/s11306-012-0413-9
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发表时间：
2012
期刊：
影响因子：
3.6
通讯作者：
Wurm M
中科院分区：
文献类型：
--
作者：
Wurm M
从受损哺乳动物细胞到周围介质的代谢物泄漏的定量对于用于代谢组学分析的样品的处理是高度感兴趣的。了解促进代谢物通过选择性透化细胞膜释放所需的典型时间跨度也是相关的。这种过程的实时观察是困难的，因为小的代谢物不能通过光学方法直接观察到，并且其他更间接的测定可以干扰代谢物浓度本身。然而，可以基于从具有已知扩散系数的易于检测的分子获得的参考测量来预测代谢物从细胞质的基于扩散的损失。在这项工作中，我们使用绿色荧光蛋白（GFP）作为标记物，并使用有限元方法模拟其从受损细胞中的释放。破裂的膜面积分数，Ad，膜破裂的分布和GFP流出率，KE之间的相关性已经建立。KE已被实验确定为中国仓鼠卵巢细胞，这已被机械损坏的通道通过微喷嘴的几何形状在微流控系统中。已经实时观察到微喷嘴下游的即时GFP释放，并且已经预测了相应的膜损伤。在此基础上，我们计算了自由扩散的细胞溶质葡萄糖的排水所需的预期时间，发现对于1.5%的破坏膜面积分数，在1 s内损失了1.90%。因此，即使是最小的膜损伤也会导致细胞溶质代谢物通过扩散快速损失，除非发生膜重新密封过程。
The quantification of metabolite leakage from damaged mammalian cells to the surrounding medium is of high interest for the processing of samples for metabolomic analysis. It is also of relevance to know the typical time span which is required for a promoted metabolite release through a selectively permeabilized cell membrane. The real-time observation of such a process is difficult since small metabolites cannot be observed directly by optical methods and other more indirect assays can disturb the metabolite concentration itself. However, the diffusion based loss of metabolites from the cytoplasm can be predicted on the basis of reference measurements taken from an easy-to-detect molecule with known diffusion coefficient. In this work, we use green fluorescent protein (GFP) as a marker and model its release from damaged cells using the finite-element method. A correlation between the disrupted membrane area fraction,Ad, the distribution of membrane ruptures and the rate of GFP efflux,ke, has been established.kehas been determined experimentally for Chinese hamster ovary cells, which have been damaged mechanically by passage through a micronozzle geometry in a microfluidic system. The immediate GFP release downstream of the micronozzles has been observed in real-time and the corresponding membrane damage has been predicted. On this basis, we calculated the expected times required for the drainage of freely diffusable cytosolic glucose and found a loss of ≈90% within 1 s for a disrupted membrane area fraction of ≈5%. Hence, even minimal membrane damage would lead to a rapid loss of cytosolic metabolites by diffusion unless membrane resealing processes take place.
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影响因子：
3.3
作者：
Togo, T;Krasieva, TB;Steinhardt, RA
通讯作者：
Steinhardt, RA
影响因子：
21.8
作者：
Kuimova, Marina K.;Botchway, Stanley W.;Ogilby, Peter R.
通讯作者：
Ogilby, Peter R.
用于悬浮动物细胞代谢组采样的快速过滤
影响因子：
2.7
作者：
M. Volmer;Stefan Northoff;S. Scholz;T. Thüte;H. Büntemeyer;T. Noll
通讯作者：
T. Noll
影响因子：
3.4
作者：
Swaminathan, R;Hoang, CP;Verkman, AS
通讯作者：
Verkman, AS
DOI：
10.1002/pol.1963.100010218
发表时间：
1963
期刊：
Journal of Polymer Science Part A
影响因子：
--
作者：
M. Bourne;C. Chichester;C. Sterling
通讯作者：
C. Sterling