Introduction of Macromolecules into Living Dictyostelium Cells by Electroporation.

概要

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An attempt was made to optimize conditions for introduction of macromolecules into Dictyostelium cells by electroporation. The amount of fluorescein-labeled bovine serum albumin (FITC-BSA) introduced into cells was measured by fluorometry after extraction of FITC-BSA from cells with detergent. The amount increased as the applied voltage and capacitance of the discharger were increased. However, the survival of cells decreased at higher voltages and elevated capacitance. FITC-BSA was introduced into 80-90% of treated cells. FITC-BSA at 0.25mg/ml was introduced into cells under optimum conditions when the concentration of the extracellular protein was 2.5mg/ml. Several discharges in sequence improved the extent of introduction of FITC-BSA although viability decreased. There was a linear correlation between final intracellular concentration and the initial extracellular concentration of FITC-BSA, suggesting the possible quantitative introduction of the protein into cells. The membrane pores that opened during electroporation closed within 2.5 sec after the discharge. FITC-labeled dextran with molecular weights of less than 5×105 were able to pass through these pores. Our results show that electroporation provides a quantitative and reproducible method for introduction of macromolecules into living Dictyostelium cells.
日本細胞生物学会の論文