Development of cisplatin‐resistant cell lines

Parental A549, H157, and IL‐6 knockdown A549IL‐6si/sc, and H157IL‐6si/sc cells were continuously treated with gradually increased doses of cisplatin for 6 months according to the method described by Barr et al.¹¹ Briefly, cells were treated with 1 μM cisplatin for 72 h and allowed to recover for the following 72 h. After repeating one more cycle at 1 μM cisplatin concentration, the cells were then treated with 2 μM cisplatin in the following two cycles. This procedure was continued with increasing cisplatin concentration up to 30 μM. During the cisplatin‐resistance induction procedure, the IC₅₀ values of every five passage cells were accessed in comparison with those of the parental cells until the IC₅₀ value remained constant. The IC₅₀ values were calculated using GraphPad Prism 5.0 software (San Diego, CA, USA). The cisplatin‐resistant cell lines obtained by this method were maintained in growth media containing 10 μM cisplatin.