Measurement of intracellular copper level

Three experimental groups were designed, namely CuCl₂ group, CuET group and CuET (tBHP) group. Cells in the CuET (tBHP) group were pretreated with 2.5 mM tBHP (Sigma, 458139) for 2 h, which was reported to increase the intracellular ROS level by 40%²¹. The other two groups of cells were pretreated with ddH₂O. After pretreatment, the three groups of cells were treated with 5 μM CuCl₂, 5 μM CuET and 5 μM CuET for 3 h, respectively. Then, cells were harvested using trypsin-EDTA solution, and washed twice with metal-free Hanks’ balanced salt solution (HBSS). Next, 300 μL lysis buffer (Beyotime, P0013) was added to each group of cell pellets. Aliquots of the three cell suspensions were collected to measure the protein content. Finally, cell suspensions were digested with 5 mL concentrated nitric acid and then analyzed for copper content using ICP-MS.