Ecm 830 square wave electroporation system

GMC suspensions were electroporated in single cuvettes containing pre-warmed culture medium and a pool of 2 target-specific TRPC6 siRNAs or a non-targeting control siRNA (Sigma-Aldrich, St. Louis, MO). An electrical field to induce siRNA transfection was applied using the ECM 830 square wave electroporation system (Harvard Apparatus, Holliston, MA). Ten minutes after electroporation, the cells were plated for ~72 hours. Western immunoblotting was used to confirm efficient knockdown. GFP-tagged human nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1; OriGene Technologies, Rockville, MD) cDNA clone (400 ng/mL) was transfected into GMCs using the TurboFect transfection reagent (Life Technologies, Grand Island, NY). Transfected cells were maintained in 5% CO₂ humidified incubator for 48 h at 37 °C. Images were acquired using a Zeiss laser-scanning confocal microscope.

All procedures were approved by the University of South Florida Institutional Animal Care and Use Committee (protocol R2736, 2005). One million melanoma cells in 50 µL phosphate-buffered saline were injected subcutaneously in the left flank of female 7-to-8-week-old C57Bl/6J mice (Jackson Laboratories, Bar Harbor, ME, USA). Tumors were allowed to grow for eight days to a diameter of approximately 4 mm. Mice with tumors were randomized into groups then anesthetized using a mixture of 2.5% isoflurane and 97.5% O₂. Four groups of four included mice with control untreated tumors, tumors injected with 50 µg plasmid DNA in 25 µL sterile physiological saline, tumors injected with 25 µL saline followed by pulse application, and tumors injected with plasmid DNA followed by pulse application. A 6-needle array controlled by an autoswitcher was fitted around the tumor, and delivery was immediately performed by the application of six 100 μs pulses with a voltage-to-distance ratio of 1300 V/cm and a frequency of 4 hertz using a legacy model ECM 830 Square Wave Electroporation System (BTX Harvard Apparatus, Holliston, MA, USA). Each mouse was monitored continuously until recovered from anesthesia, as indicated by their ability to maintain sternal recumbency and exhibit purposeful movement. Mice were humanely euthanized after four hours. Then, tumors were removed and snap frozen on dry ice.

Anesthesia, aseptic technique, perioperative care, and analgesia were performed in accordance with approved standard procedures and protocols. Anesthesia and analgesia consisted of 2% inhaled isoflurane, 0.05–0.1 mg/kg buprenophine, and 5-10 mg/kg meloxicam. A 1 cm midline incision was made directly below the xiphoid and gentle pressure was applied on both sides of the incision to expose the left lobe of the liver. IRE was performed by gently holding the lateral half of the left lobe between custom 10 mm-diameter circular-plate copper electrodes that were affixed to calipers and connected to an ECM 830 Square Wave Electroporation System (BTX Harvard Apparatus, Holliston, MA). The thickness of the liver lobe of each mouse was measured before IRE administration to determine the voltage required to deliver the prescribed electric field strength. Electrical pulses were applied using parameters of 1500 V/cm, 8 total 100 μs square pulses, each pulse separated by 100 ms. After IRE, the liver lobe was returned to the abdomen, and the abdominal incision was closed by sutures in two layers.