A novel approach for monitoring extracellular acidification rates: based on bead injection spectrophotometry and the lab-on-valve system.
A novel approach for monitoring extracellular acidification rates: based on bead injection spectrophotometry and the lab-on-valve system.
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DOI：
10.1039/b315007k
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发表时间：
2004-02
期刊：
影响因子：
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通讯作者：
H. Erxleben;M. Manion;D. Hockenbery;L. Scampavia;J. Ruzicka
中科院分区：
文献类型：
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作者：
H. Erxleben;M. Manion;D. Hockenbery;L. Scampavia;J. Ruzicka
监测细胞外酸化速率（ECARs）对于研究细胞活动非常重要，因为它可以评估改变代谢功能的因素，如兴奋剂、抑制剂、毒素以及受体和非受体介导的事件。虽然光可寻位电位传感器（细胞传感器微生理计）在过去一直是ECARs测量的主要工具，但这项工作引入了一种新方法，该方法利用微载体珠（Sephadex）表面固定的pH指示剂，并用光纤耦合分光光度计进行探测。同样，被研究的活细胞也被固定在微载体珠（Cytopore）上。这些珠子在一个被称为阀上实验室（LOV）的微流体系统中被测量、运输和监测。将载珠与珠注分光光度法和阀上实验室模块（BIS-LOV）结合使用，使ECAR测量可靠且自动化。BIS-LOV方法的可行性被证明是通过测量小鼠肝细胞系TABX.2S的ecar来实现的，该细胞系生长在LOV系统中央通道内的Cytopore珠上。将这些固定的细胞灌注在磷酸盐缓冲载体溶液中（容量：1 mmol L(-1), pH 7.4）。从10(5)到10(6)个细胞中挤出的质子在220秒的停止流动期间积累，随后测量pH值，通过与微载体珠结合的pH指示剂的吸光度变化来检测。在载体缓冲液中加入代谢抑制剂（叠氮化钠、肟酸），可以以非常可重复的方式诱导基础质子挤出率的增加或减少。BIS-LOV技术与细胞传感器微生理仪和文献的比较证实了这种新方法的有效性，突出了它的优势，并提出了未来的改进，将使BIS-LOV成为常规ECARs测量的实用工具。
Monitoring extracellular acidification rates (ECARs) is important for the study of cellular activities, since it allows for the evaluation of factors that alter metabolic function, such as stimulants, inhibitors, toxins as well as receptor and non-receptor mediated events. While the light addressable potentiometric sensor (Cytosensor Microphysiometer) has been the principal tool for ECARs measurement in the past, this work introduces a novel method that exploits an immobilized pH indicator on the surface of microcarrier beads (Sephadex) and is probed with a fiber optic coupled spectrophotometer. Likewise, live cells under investigation were also immobilized on microcarrier beads (Cytopore). These beads are metered, transported and monitored within a microfluidic system, termed as the Lab-on-Valve (LOV). Use of carrier beads in conjunction with Bead Injection Spectrophotometry and a Lab-on-Valve module (BIS-LOV), makes ECAR measurements reliable and automated. The feasibility of the BIS-LOV approach is demonstrated measuring ECARs of the mouse hepatocyte cell line, TABX.2S, grown on Cytopore beads packed within the central channel of the LOV system. These immobilized cells were perfused in a phosphate buffer carrier solution (capacity: 1 mmol L(-1), pH 7.4). Protons extruded from 10(5) to 10(6) cells were accumulated during a stopped flow period of 220 s followed by a pH measurement, detected by changes in absorbance of the pH indicator bonded to the microcarrier beads. Addition of metabolic inhibitors (sodium azide, oxamic acid) to the carrier buffer solution can induced an increase or decrease of the basal proton extrusion rate in a very reproducible manner. Comparison of the BIS-LOV technique to the Cytosensor microphysiometer and literature confirms the validity of this novel approach, highlighting its advantages and suggesting future improvements that will make the BIS-LOV a practical tool for routine ECARs measurement.