Electroporation Instrumentation. For electroporation experiments, cell dishes were mounted in a circular polycarbonate holder by using vacuum grease and were transferred to the stage of an inverted microscope (Leica DM IRB, Wetzlar, Germany) equipped with a Leica PL Fluotar 40× objective. A cell was selected, and two carbon fiber microelectrodes (ProCFE, Axon Instruments, Foster City, CA) with an outer diameter of 5 µm were positioned close at each side of the cell by high-graduation micromanipulators (Narishige MWH-3, Tokyo). The two carbon fiber electrode tips (anode and cathode) were positioned 2 to 5 µm from the boundaries of a cell soma or cellular process at an angle of 0-20° and 160-180° with respect to the object plane. The planar electrode arrangement, as schematically shown in Fig. 1, can be used because the electrode tips are flexible and sustain the mechanical stress of being forced against the coverslip. This configuration was, however, only used on sparse cultures of newly seeded cells essentially lacking processes. It is important to not place the electrodes in direct contact with the cell to prevent electrochemical transformation of proteins and other membrane-associated structures. Cells were permeabilized with multiple 1-ms pulses (except in the case of plasmid introduction in which single pulse protocols were used) by using a pulse generator (Digitimer Stimulator DS9A, Welwyn Garden City, U.K.). The voltage output from the pulse generator was calibrated by using high-impedance electrodes and a patch-clamp amplifier. Because the cells sometimes had complex geometries, the membrane voltages reported were obtained by multiplying the applied electric field strength with the average radius from the boundary of the anode-facing cell membrane to the boundary of the cathode-facing membrane at the center of the electrodes.
The electroporation buffer in the fluorescein experiments was a Hanks'-Hepes solution (137 mM NaCl/5.4 mM KCl/0.41 mM MgSO4/0.4 mM MgCl2/1.26 mM CaCl2/0.64 mM KH2PO4/3.0 mM NaHCO3/5.5 mM D-glucose/20 mM Hepes, pH adjusted to 7.4 with NaOH) supplemented with 5 to 10 µM fluorescein. A Ca2+-free Hanks'-Hepes solution containing 10 µM fluo-3 and 70 µM EDTA was used in the fluo-3 experiments. For cDNA transfection experiments, a Hepes buffer solution (140 mM NaCl/0.75% dimethyl sulfoxide/10 mM Hepes, pH adjusted to 7.4 with NaOH) containing 10 µg plasmid/ml was used. For electroporation of individual cellular processes, cells first were incubated for 10-30 s in a thapsigargin-supplemented (500 nM) Hanks'-Hepes solution, and then electroporation was performed in a Ca2+-free Hanks'-Hepes solution containing 10 µM fluo-3 and 70 µM EDTA. For selective staining of mitochondria, cells were incubated for 20 min in a rhodamine 123-supplemented (10 µM) Hanks'-Hepes solution and were rinsed with buffer solution before viewed in the microscope.
Fluorescence imaging was achieved by sending the output of an argon ion laser (Spectra-Physics 2025-05, 488 nm) through a 488-nm line interference filter followed by a spinning disk to break the coherence and scatter the laser light. The laser light was collected by a lens and was sent through a fluorescein filter cube (Leica I-3) into the objective to excite the fluorophores. The resulting fluorescence was collected by the same objective, and the image was detected by a three-chip color charge-coupled device camera (Hamamatsu, Kista, Sweden) and was recorded at a 25-Hz frame collection rate by a Super VHS (Panasonic S-VHS AG-5700, Stockholm, Sweden). The charge-coupled device images were digitized from tape and were processed for presentation. RGB (Red Green Blue) files were converted to CMYK (Cyano Magenta Yellow Black) file format for printing. Occasionally, the charge-coupled device camera was fed directly into a frame grabber.
Cell Culture. Progenitor cells derived from adult rat brain hippocampus were cultured according to procedures described by Palmer et al. (24) and were plated onto no. 1, 1-inch circular coverslips coated with polyornithine and laminin as described by Ray et al. (25). COS 7 cells derived from African green monkey kidneys, and 293 cells were cultured according to standard procedures.
Chemicals and Materials. Hepes (>99%), sodium chloride, potassium chloride, and sodium hydroxide (all Suprapur), calcium dichloride, magnesium dichloride, magnesium sulfate, potassium dihydrogen phosphate, sodium hydrocarbonate, and EGTA (Titriplex VI) (all pro analysi) were purchased from Merck. D-glucose (AnalaR) was from BDH and fluorescein (GC-grade) and dimethyl sulfoxide were obtained from Sigma. Fluo-3 (pentaammonium salt), rhodamine 123 and thapsigargin were from Molecular Probes, and the plasmid pRAY 1 was from GIBCO/BRL. Deionized water from a Milli-Q system (Millipore) was used.
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