DETERMINATION OF CELL ELECTROPORATION IN SMALL-VOLUME SAMPLES

摘要

Expose of cells to electric field pulses increases the cell membrane permeability. Intracellular potassium ions leak out of the cells through aqueous pores created in the membrane. This release is used here for the determination of the fraction of electroporated cells. To determine cell membrane electroporation in small-volume samples (40-50μl), mini both potassium ion-selective and reference electrodes, with tip diameters of 1-1.5mm and minimum immersion depths of 1mm, were utilized. The obtained calibration graph was linear within the concentration range 0.2-100mM. The slope was 50-51 and 53-56mV per concentration order at 10-11 and 19-21°C, respectively. Detection limit of the electrode was determined to be 0.05-0.08mM, however, it was possible to work down to concentrations in the range of 0.01mM. Experiments have been carried out on human erythrocytes exposed to a square-wave electric pulse with the duration of 0.1-2ms. The extracellular potassium concentrations were in the range between 0.04-0.08mM (intact cells) and 3-5mM (100% electroporation). The obtained dependences of the fraction of electroporated cells on the pulse intensity were of a sigmoid shape. The dependence of the pulse amplitude required to electroporate 50% of cells on the pulse duration, obtained from the release of intracellular potassium ions, coincided with the one determined from the extent of hemolysis after 24 h-incubation at low temperature.