DELIVERY OF MACROMOLECULES INTO ADHERENT CELLS VIA ELECTROPORATION FOR USE IN FLUORESCENCE SPECTROSCOPIC IMAGING AND METABOLIC STUDIES

A method is described to introduce by electroporation membrane-imperme ant molecules into adherent Living cells with little perturbation, The approach uses simple, commonly available equipment to introduce small fluorescent dyes, large carrier-based dyes (e.g,, fluorescein-labeled dextran), large macromolecules (e.g,, antibodies), and metabolic prec ursors (e.g., P-32-ATP) with high efficiency. Conditions are relativel y independent of cell type, Electroporation with three pulses of 300 v olts at 540 mu F capacitance at 4 degrees C is a good starting point f or many cell types. Electrode distance from the adherent cells was cri tical at 1.0 +/- 0.15 mm. Suitable poration medium includes calcium-ma gnesium free phosphate buffered saline (PBS), PBS-buffered 0.25-3.0 M sucrose, Hepes-buffered sucrose, or unbuffered sucrose, Potential use in fluorescence imaging and metabolic studies is shown with DNA synthe sis, cell replication, cell substratum attachment, P-32-ATP phosphoryl ation, and insulin-mediated increases in glucose uptake and its suppre ssion by antiphosphotyrosine and antiglucose transporter protein antib odies, The ability to load foreign molecules into large numbers of adh erent cells provides a means of studying these cells individually via microscopic approaches, such as fluorescence spectroscopic Imaging, as well as with conventional biochemical and physiological techniques. ( C) 1996 Wiley-Liss, Inc.