Serial replating colony-forming assay

Primary human AML cells were cultured at 2.5 × 10⁴/mL in the presence of various drugs in MethoCult H4434 (Stem Cell Technologies). Dishes were incubated at 37°C in 5% CO₂ with ≥95% humidity for 10 to 14 days to allow for colony formation. Colonies were counted and recorded; the cells were rinsed with prewarmed PBS (37°C), counted, and replated in fresh medium at a concentration of 2.5 × 10⁴/mL, to test whether they would form secondary colonies.