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研究目的（著者による）
To determine adequate experimental conditions that allow electropermeabilization without damage to embryonic amphibian cells.
詳細情報
The technique of electropulsation has been shown to be highly efficient in promoting penetration of exogenous molecules into living cells, transfection, and cell fusion in different vegetal, animal, and bacterial cell systems. Introduction of such exogenous compounds, i.e., plasmids, into living cells is of interest for embryological studies. Embryonic amphibian ectodermal cells from Pleurodeles waltl gastrulae were used.
electropulsator connected to a pair of parallel stainless steel electrodes
ばく露装置の詳細
The electrodes (5 or 10 mm apart) were dipped in half diluted Holtfreter's solution used as pulsating medium (1 mm thick, 4°C) and seeded against the bottom of the culture dish.
細胞機能: cell permeabilization (cell ATP leakage into the medium (luciferase assay); uptake of exogenous fluorescent dye, pyranin, into the cells); behavior of cell culture (cell differentiation)
The data demonstrated that embryonic gastrula cells from the amphibian P. waltl, either freshly dissociated or cultured for 5 days, could be permeabilized when submitted to an external electric field using high-voltage square wave pulses: 500 V/cm for isolated spherical cells and 150-200 V/cm for plated cultured cells. With the application of higher field intensities (600 V/cm for freshly dissociated cells and 300 V/cm for cultured cells) cell fusion and syncytial structures could also be obtained. Isolated spherical cells had 100% viability immediately after being pulsed at intensities up to 600 V/cm. Cell lysis was revealed above this value, although the nonlysed cells were observed to spread on a substrate and differentiate normally. For the cultured plated cells, cell viability fell with increasing electric-field strength. Nevertheless, for electric-field intensities less than or equal to 300 V/cm, 100% of the cells remained attached to the substrate and differentiated normally over the following 5 days.
