Propidium

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.

At the end of treatment, the cells were washed and resuspended in binding buffer (Annexin V PE Apoptosis Detection kit; BD Biosciences, East Rutherford, NJ). After incubation with annexin V-PE and propidium iodine (PI), binding buffer was added, and the cells were analyzed via fluorescence-activated cell sorting (FACScan, BD Biosciences). Data analysis was performed using Cell Quest software.

Tunnel assay was performed according to DeadEnd™ Fluorometric TUNEL System (Promaga, Tokyo, Japan) manufacturer’s instructions. Briefly, tumor sections were treated with fresh xylene in a Coplin jar for 5 min twice at room temperature. Rehydrate the samples by sequentially immersing the slides through graded washes (100%, 95%, 85%, 70%, 50%, 0.85% NaCl, PBS) for 5 min. The tissue sections were fixed by immersing the slides in 4% methanol-free formaldehyde solution in PBS for 15 min. After washed in PBS for 5 min three times, the samples were incubated with 100 µl proteinase K (20 µg/ml) for 8–10 min. Then were washed and incubated with 100 µl Equilibration Buffer for 5-10 min. The tissue sections were fixed with 4% methanol-free formaldehyde solution in PBS for 5 min. Then washed and incubated with 50 µl of rTdT incubation buffer at 37 °C for 60 min in the dark. The reactions were terminated by incubated with 2X SSC for 15 min at room temperature. The samples were washed three times and were stained by propidium (1 µg/ml) in PBS for 15 min at room temperature in the dark. Then the samples were washed three times and analyzed by the Olympus FV1000 IX81-SIM Confocal Microscope (Olympus, Tokyo, Japan).

The cells were digested into a single-cell suspension, washed with 70% alcohol, then fixed for 4 h to overnight at 4 °C. Afterward, appropriate amounts of RNase A (KeyGEN BioTECH, Nanjing, China) and propidium (PI) (KeyGEN BioTECH, Nanjing, China) were added to the cell suspension. The cells were incubated at 37 °C for 30 min in the dark. Flow cytometry (BD FACSCalibur; BD Biosciences, NJ, USA) was then used to analyze the cells.