ABSTRACT
Dielectrophoresis (DEP)--the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K(+) and Ca(2+) levels were extremely similar between sensitive and resistant lines, levels of Cl(-) were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function.
Subject(s)
Cell Membrane/drug effects , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Electrophoresis/methods , Ion Channel Gating/drug effects , Ion Channels/antagonists & inhibitors , Membrane Transport Modulators/administration & dosage , Humans , K562 Cells , VerapamilABSTRACT
A type of well-based assay that uses a laminated three-dimensional electrode design to characterise the effects of different drugs on red blood cells using dielectrophoresis is presented. The capability of the system to determine the effects of chemical agents on the electrophysiology of red blood cells is demonstrated using saponin and valinomycin as two examples of drugs that can penetrate the cell membrane and therefore change the dielectric properties of the cell. Light intensity changes are measured in the well over a period of time at various frequencies and the dielectric properties of the cells determined using an ellipsoidal multi-shell model. It is shown that the laminated electrode permits a high degree of automation and thus a high number of parallel experiments, which reduces both the time and effort needed to examine differences between populations of red blood cells. The technique is directly compatible with the industry-standard 1536 well-plate analysis technique.