Establishment of TMZ-resistant glioma cell lines

1×10⁵ U251 or LN229 cells were exposed to an initial TMZ concentration of 1 µM for 15 days. The surviving population of cells was grown to 80% confluence and passaged twice over 3 weeks. The concentration of TMZ was then sequentially increased in the same manner to 5 µM for 15 days, 25 µM for 15 days, 50 µM for 15 days, 100 µM for 20 days, 200 µM for 20 days, 500 µM for 25 days, 1,000 µM for 25 days and finally to the concentration of 2,000 µM for the last 30 days. The half maximal inhibitory concentration (IC₅₀) of U251 cell was 1,093 µM, while the IC₅₀ of TMZ-resistant U251 cell was 1,722 µM. The IC₅₀ of LN229 cell was 1,018 µM, while the IC₅₀ of TMZ-resistant LN229 cell was 1,571 µM. The established TMZ-resistant U251 or LN229 cells were designated as U251^TMZ or LN229^TMZ, respectively.