MTT assay

MTT assay was performed to determine the cytotoxicity of direct and indirect plasma treatments and to find the optimum doses. B₁₆F₁₀ melanoma cells were cultured at density of 4 × 10⁴ cells per well in the complete medium. Cells’ conditioned medium was removed after 24 hours and CAP irradiated in the direct and indirect methods as follows.

In the direct method, a complete medium was added to the cells and then they were treated by CAP for different dosages of 0, 2, 4 and 6 minutes.

In the indirect method, 1 mL a complete medium was transferred to a 6-well plate, and was treated by CAP for 0, 2, 4, and 6 minutes. Then the plasma activated medium was added to each of the 6-well plates.

Direct and indirect treatments were compared and the optimum dosage of plasma was determined, second comparison was conducted to investigate the effect of combination of indirect plasma and common therapies such as chemical drug. In the direct treatment, cell supernatant culture medium was replaced with 1.5 ml of complete medium and then CAP was imposed for 6 minutes.

In indirect treatment, 1.5 ml of completed medium was treated by CAP in 6-well plate for 6 minutes; afterwards the complete activated medium was transferred on the cells. In chemotherapy group, cells received 500 μg/ml of cyclophosphamide and in a combination of chemotherapy and indirect CAP treatment group cells received 500 μg/ml of cyclophosphamide and also received 1.5 ml plasma activated medium.