Cell Culture and PEF Cytotoxicity in 3D Cultures

Three-dimensional cultures in agarose were described in detail previously.³⁷ Briefly, the bottom of a 35-mm dish was coated with 3 mL of 2.5% low-gelling-temperature agarose (Sigma-Aldrich, St Louis, Missouri) in the growth medium. Cells were resuspended at 5 × 10⁶ cell/mL in 1% agarose in the growth medium, and 1.5 mL of this suspension was pored over the presolidified 2.5% agarose base layer. The samples were incubated at 4°C for 5 minutes to speed up agarose jellification thus avoiding cell sedimentation, covered with 0.5 mL of media, and kept in the incubator for 30 minutes before PEF treatment.

The agarose cultures were analyzed 2 hours after exposure. Dead cells were stained using 4 μg/mL of propidium iodide (PI; Sigma-Aldrich) in phosphate-buffered saline (PBS). Thirty minutes before the analysis, the growth medium was replaced with 1 mL of PI solution. Images of the ablation zone were acquired using an Olympus SZX16 fluorescence stereo microscope (Olympus America, Hamden, Connecticut) equipped with a Hamamatsu C9100 EM-CCD camera using a 0.9×, 0.44 NA objective.

Images were analyzed with MetaMorph version 7.5.2 software (Molecular Devices, Foster City, California). Propidium (Pr) signal was quantified in the center of the gap between the electrodes within a region of 1 mm² with almost uniform electric field (Figure 2A). For each image, the fluorescence intensity of the exposed area was corrected for the background fluorescence.