Gwiz luc

Endotoxin-free plasmid DNA encoding luciferase, gWizLuc (size, 6,732 bps), was purchased from Aldevron (Fargo, ND). Plasmid DNA was suspended in sterile saline at 2 mg/mL. To reduce the number of animals in the study, we utilized 6 treatment sites on each flank of the guinea pig. There was a 1–2 cm gap between treatment sites, which was maintained to avoid any treatment interference between sites. There was a total of 24 treatment sites which were assigned to six groups with n = 4 sites in each group. The six treatment groups consisted of the control (injection only), gene electrotransfer (GET) only, plasma treatment only for 1 min or 3 min, and combined treatment groups: plasma for 1 min or 3 min plus GET. Each treatment site received a 50 μL injection of gWizLuc at a concentration of 2 mg/mL. For the plasma treatment, the gap distance between the plasma nozzle and the skin surface was kept constant at 10 mm. For the GET treatment, a multi electrode array (MEA) containing 16 spring-loaded pins serving as non-penetrating electrodes, configured in a 4 × 4 array and equally spaced for a 2 mm – interelectrode distance[27 (link), 28 (link), 47 (link)]. Only 4 electrodes were active at a time. Each set of 4 electrodes delivered 4 pulses and nine sequences were used to sequentially activate all 16 electrodes (4 electrodes at a time)[27 (link)]. Total of 72 pulses were applied for each GET treatment. The electric pulse parameters were 150 ms-duration, 35 V pulses at a pulse repetition rate of 6.7 Hz (or 150 ms pulse-to-pulse interval). The electric pulses were generated using a high voltage power supply (Ultravolt HV Rack 1/4C24-P250, Ronkonkoma, NY) that was controlled by a customized program utilizing LabVIEW 8.2.1(National Instruments, Austin, TX). Sites that were assigned to the combined treatment groups were exposed to plasma for 1 min or 3 min, and immediately subjected to the pulsed GET treatment.

gWiz plasmid containing luciferase (gWiz-Luc) and plasmid DNA encoding BDNF (gWiz-BDNF) were purchased from Aldevron (Fargo, ND). Collagen type I was purchased from Corning (Discovery Labware Inc., Bedford, MA) and endothelial cell basal medium-2 (EBM-2) was procured from Lonza (Walkersville, MD). Hydrocortisone, human basic fibroblast growth factor, and ascorbic acid were purchased from Sigma-Aldrich (Saint Louis, MO). Dithiothreitol (DTT), glycylglycine, ethylenediaminetetraacetic acid (EDTA), HEPES, and magnesium chloride hexahydrate (MgCl₂.6H2O) were received from Fisher Scientific (Pittsburgh, PA). Aprotinin solution was purchased from Fisher Bioreagents, New Zealand. The penicillin-streptomycin solution, Lipofectamine 3000 transfection reagent, and chemically defined lipid concentrate were procured from Invitrogen (Carlsbad, CA). Heat inactivated fetal bovine serum was bought from Hyclone Laboratories (Logan, UT). Pierce MicroBCA and BCA protein assay kits were purchased from Thermo Scientific (Rockford, IL). Quant-iT PicoGreen dsDNA Assay kit was obtained from Molecular Probes, Inc. (Eugene, OR). CellTiter-Glo 2.0 reagent (ATP assay), Beetle Luciferin, potassium salt, and cell culture lysis 5× Reagent were purchased from Promega (Madison, WI). Laemmli sodium dodecyl sulfate (SDS) sample buffer and RIPA buffer, 5×, were procured from Alfa Aesar (Ward Hill, MA). Adenosine-5-triphosphate (ATP) was obtained from MP Biomedicals (Illkirch, France). Odyssey blocking buffer was purchased from Li-COR (Lincoln, NE). Mouse monoclonal antibody to GRP94 and Alix were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Mouse monoclonal antibodies against ATP5A and GAPDH were procured from Abcam. Mouse monoclonal antibody against CD9 was obtained from Life Technologies Corporation, whereas Alexa Fluor 790-conjugated donkey anti-mouse IgG was received from Jackson ImmunoResearch Lab Inc. (West Grove, PA). Calcein AM, flow cytometry sub-micron particle size reference kit, and an Attune NxT small particle side scatter filter were purchased from Invitrogen (Carlsbad, CA).