Control of the release of freely diffusing molecules in single-cell electroporation.
Single-cell electroporation using an electrolyte-filled capillary is an emerging technique for transient pore formation in adherent cells. Because adherent cells do not have a simple and consistent shape and because the electric field emanating from the tip of the capillary is inhomogeneous, the Schwan equation based on spherical cells in homogeneous electrical fields does not apply. We sought to determine experimental and cell parameters that influence the outcome of a single-cell electroporation experiment. A549 cells were exposed to the thiol-reactive dye Thioglo-1, leading to green fluorescence from intracellular thiol adducts. Electroporation causes a decrease with time of the intracellular fluorescence intensity of Thioglo-1-loaded cells from diffusive loss of thiol adducts. The transient curves thus obtained are well-described by a simple model originally developed by Puc et al. We find that the final fluorescence following electroporation is related to the capillary tip-to-cell distance and cell size (specifically, 2(A/pi)(1/2) where A is the area of the cell's image in pixels. This quantity is the diameter if the image is a circle). In separate experiments, the relationship obtained can be used to control the final fluorescence following electroporation by adjusting the tip-to-cell distance based on cell size. The relationship was applied successfully to A549 as well as DU 145 and PC-3 cells. Finally, F-tests show that the variability in the final fluorescence (following electroporation) is decreased when the tip-to-cell distance is controlled according to the derived relationship in comparison to experiments in which the tip-cell distance is a constant irrespective of cell size.
Agarwal
Aparna
A
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Wang
Manyan
M
Olofsson
Jessica
J
Orwar
Owe
O
Weber
Stephen G
SG
D002021:Buffers; D045744:Cell Line, Tumor; D020552:Comet Assay / Q000379:methods*; D004058:Diffusion; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009281:Naphthalenes / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*
D002021:Buffers; D005456:Fluorescent Dyes; D009281:Naphthalenes; D011758:Pyrroles; C498335:ThioGlo-1