The procedures were similar to those described recently [11] (link), [53] (link). For the passage immediately preceding experiments, CHO cells were transferred onto glass cover slips pre-treated with poly-L-lysine to improve cell adhesion. After several hours, a cover slip with cells was transferred into a glass-bottomed chamber (Warner Instruments, Hamden, CT) mounted on an Olympus IX71 inverted microscope equipped with an FV 300 confocal laser scanning system (Olympus America, Center Valley, PA). The chamber was filled with a buffer composed of (in mM): 136 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 10 Glucose (pH 7.4), with addition of 30 µg/ml of propidium iodide. The buffer osmolality was at 290–300 mOsm/kg, as measured with a freezing point microosmometer (Advanced Instruments, Inc., Norwood, MA). The chemicals were purchased from Sigma–Aldrich.
EPs were delivered to a selected cell (or a group of 2–4 cells) with a pair of tungsten rod electrodes (0.08-mm diameter, 0.15 mm gap). With a help of a robotic micromanipulator (MP-225, Sutter, Novato, CA), these electrodes were positioned precisely at 50 µm above the coverslip surface so that the selected cells were in the middle of the gap between their tips. Nearly rectangular 60-ns pulses were generated in a transmission line-type circuit, by closing a MOSFET switch upon a timed delivery of a TTL trigger pulse from pClamp software via a Digidata 1322A output (MDS, Foster City, CA). The exact PRF, the EP delivery protocol, and synchronization of EP delivery with image acquisitions were programmed in pClamp.
The E-field between the electrodes was determined by 3D simulations with a finite element Maxwell equations solver Amaze 3D (Field Precision, Albuquerque, NM). The exact EP shapes and amplitudes were captured and measured with a Tektronix TDS 3052 oscilloscope.
Differential-interference contrast and fluorescent images of cells (excitation: 488 nm; emission 605 nm) were collected every 10 sec (starting exactly 50 s prior to the first EP) using a 60x, 1.42 NA oil objective. Photomultiplier tube settings were biased towards high sensitivity and detection of even minimal propidium uptake, although massive uptake caused detector saturation. Images were quantified with MetaMorph v. 7.5 (MDS). All experiments were performed at a room temperature of 22–24°C.
EPs were delivered to a selected cell (or a group of 2–4 cells) with a pair of tungsten rod electrodes (0.08-mm diameter, 0.15 mm gap). With a help of a robotic micromanipulator (MP-225, Sutter, Novato, CA), these electrodes were positioned precisely at 50 µm above the coverslip surface so that the selected cells were in the middle of the gap between their tips. Nearly rectangular 60-ns pulses were generated in a transmission line-type circuit, by closing a MOSFET switch upon a timed delivery of a TTL trigger pulse from pClamp software via a Digidata 1322A output (MDS, Foster City, CA). The exact PRF, the EP delivery protocol, and synchronization of EP delivery with image acquisitions were programmed in pClamp.
The E-field between the electrodes was determined by 3D simulations with a finite element Maxwell equations solver Amaze 3D (Field Precision, Albuquerque, NM). The exact EP shapes and amplitudes were captured and measured with a Tektronix TDS 3052 oscilloscope.
Differential-interference contrast and fluorescent images of cells (excitation: 488 nm; emission 605 nm) were collected every 10 sec (starting exactly 50 s prior to the first EP) using a 60x, 1.42 NA oil objective. Photomultiplier tube settings were biased towards high sensitivity and detection of even minimal propidium uptake, although massive uptake caused detector saturation. Images were quantified with MetaMorph v. 7.5 (MDS). All experiments were performed at a room temperature of 22–24°C.
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