Colony formation assay

Colony formation assay is widely used to test the ability of a single cell to grow into a colony, which enables us to predict tumor cell sensitivity to cytostatic drugs. We performed the colony formation assay as described previously¹⁷. Briefly, GBM cells were seeded at a density of 1 × 10³ cells per well into six-well plates and were cultured at 37 °C in a humidified incubator with 5% CO₂. After colonies became visible, the medium was replaced with fresh DMEM containing vehicle (DMSO), CPX, BTZ, or a combination of CPX and BTZ. Cells were incubated for another 5–7 days and then fixed with methanol for 20 min at room temperature. Colonies were stained with 0.1% crystal violet for 20 min at room temperature and imaged and quantified using ImageJ software. The BTZ concentration used (24 nM) was bsed on the published work by Comba A and Vlachostergios PJ^18,19.