The interdependence of Calcein electroextraction and DNA electrotransfer when processes are performed simultaneously

Abstract

Electroporation, as a cell membrane permeabilization phenomenon to various molecules, occur when cells are affected with electric field. Induced molecule diffusion through electroporated membrane is termed electrotransfer. These induced processes are employed for molecule extraction from cells as well as DNA electrotranfer to the cells. However it is not much known how molecule electroextraction is affected with DNA electrotransfer when performed simultaneously. To test this we have used chinese hamster ovary (CHO) cell line as an object. For electroextraction we used calcein-AM. Once freely diffused inside the cells, calcein-AM is metabolized into calcein. Due to its hydrophilic properties calcein cannot diffuse back to the medium through cell membrane. However calcein extraction is observed after temporary compromising cell membrane with electroporation. DNA electrotransfer was performed using PGL-4 Luciferase coding plasmid. Calcein extraction was measured with flow cytometer (BD Acura-C6). Luciferase protein relative concentration was measured with microplate reader (GENios Tecan). Electroporation was performed with one HV (1400 V/cm 100 μs) pulse. Obtained results indicated an inverse dependency between DNA transfection and calcein electroextraction. When calcein extraction was measured a highly statisticaly significant increase (P<0.001) of extracted calcein was observed when there was 100 μg/ml plasmid in medium compared with electroextraction when there is no plasmid inside the medium. When DNA transfection efficiency was measured using same experiment setup an inverse results were obtained. With plasmid concentration of 100 μg/ml in cell suspension a statistical significant decrease of transfection efficiency was observed compared with electrotransfection when there is no calcein inside the cell.[...]