Introduction of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular compartments.
Access to the cell cytoplasm in viable cells may permit direct labeling or manipulation of intracellular molecules and metabolic processes. One method to gain access to the cell cytoplasm is by electroporation, a technique that transiently creates pores in cell membranes by means of applied electrical fields. We used electroporation to introduce large-molecular-mass dextrans and proteins as probes of the cytoplasmic compartment in human gingival fibroblasts. Electrical field strength and pulse decay time were optimized to obtain cellular viability greater than 80%. Analysis by confocal microscopy and by fluorescence spectrophotometry demonstrated that a large proportion of high-molecular-mass probe was membrane-bound after electroporation. Trypsinization did not affect membrane-bound FITC-dextran but eliminated protein probe incorporated into the membrane, thereby permitting measurement of only intracellular, cytoplasmic label. Proteins of up to 66 kDa were incorporated at intracellular concentrations of 10(-15) M. After electroporation under optimal conditions, incorporated anti-vimentin antibodies were capable of binding to vimentin. Cells electroporated in the presence of RNase A exhibited significant reductions of cellular RNA. Electroporation appears to be a useful approach to probe or perturb specific cellular processes by introduction of functional molecular species into the cytoplasm of viable cells.
Glogauer
M
M
Faculty of Dentistry, University of Toronto, Ontario, Canada.
McCulloch
C A
CA
D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003911:Dextrans / Q000008:administration & dosage*; D004560:Electricity; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D011506:Proteins / Q000008:administration & dosage*
D003911:Dextrans; D011506:Proteins