Chemotherapy-induced enrichment of DiI-retaining cells in vitro. (A) DiI-retaining tumor cells were enriched in vitro after treatment with 5-fluorouracil (FU). DiI-labeled CT-26 cells were cultured in complete RPMI 1640 medium and exposed to 5-FU on day 2. The old medium was changed for fresh medium without 5-FU on day 3, then 5-FU was added again on day 4. Cells were harvested on day 6 and analyzed by flow cytometry. The control group was cultured without 5-FU. (B) Persistence of cells after 5-FU treatment. The same numbers of CT-26 cells were seeded into 96-microwell plates. The 5-FU-treated group was treated as described above, and the untreated group was cultured without 5-FU. An MTT assay was performed 5 days later. (**P < 0.01, t-test) (C) Percentage of DiI-positive cells in vitro after 5-FU treatment. (**P < 0.01, t-test). Experiments were repeated twice times with similar results. Error bars represent the SD.