Tumor Induction Cells were detached by trypsinisation and collected by centrifugation. Then, cells were resuspended in physiological saline solution in concentration of 1 107 /ml for B16F10 cells and 5 106 /ml for 4T1 cells. 100 µl of cell suspension was injected subcutaneously on the right flank of the C57Bl/6 (B16F10) and Balb/c (4T1) mice YICHANG Gloves.
Calcium Electroporation in Vivo When tumors reached size of −40 mm3 (after 6 to 9 days for B16F10 melanoma and after 8–11 days for 4T1 carcinoma) PE Gloves, treatments were performed by intratumoraly injecting 40 µl of 50 mM, 168 mM or 250 mM CaCl2, respectively. In combined treatment groups, electric pulses (8 square-wave pulses, 1300 V/cm, 100 µs, 1 Hz) were delivered immediately after injection of CaCl2 solution by stainless-steel electrodes with a 6 mm distance between the electrodes. Good contact between electrodes and skin was ensured with the use of conductive gel Disposable Gloves Wholesale.
Electric pulses were generated by Electro CELL B10 HVLV pulse generator (LEROY biotech, Betatech, Saint-Orens-deGameville, France). Throughout the entire procedure mice were kept under isoflurane inhalation anesthesia. Tumor Growth Assay Mice were monitored every day and tumor growth was determined by measuring the tumor diameters every 2 to 3 days after treatment.
Three mutually orthogonal tumor diameters were measured with Vernier caliper and volume was calculated using the following formula: Mice that responded to the treatment with complete regression of the tumors were monitored for 100 days. After that, mice were re-challenged with the injection of the same type of tumor cells on the opposite flank and the outgrowth of tumors was checked.
Statistical Analysis Statistical analysis and graphical representation was performed by GraphPad Prism software (GraphPad Holdings LLC, San Diego, CA, USA). Statistical evaluation was made by one-way-analysis of variance (One-way ANOVA). A P-value of less than 0.05 was considered statisticaly significant.