The effect of the delayed addition of ATRA on cell growth and colony formation from cultured lin⁻c-kit⁺Sca-1⁺hematopoietic precursors. FACS-enriched hematopoietic precursors (lin⁻c-kit⁺Sca-1⁺) were added to wells containing media and cytokines (SCF, IL-6, IL-11, and Flt-3-ligand). At daily intervals from 0 to 7 days after culture initiation, ATRA (1 μmol/L final concentration) was added to a separate group of wells for each time point. Cells were harvested at day 8, washed, counted, and assayed for colony-forming cell activity. The results from two separate experiments are shown. Experiment no. 1 was initiated at 50 cells/well in 96-well plates (200 μL media/well), and experiment no. 2 was initiated at 1,000 cells/well in 24-well plates (1 mL media/well). Cell counts (A and B) are shown as total cell number per well for each experiment. Colony output, CFU-GM (C and D) represent the total number of CFU-GM colonies per 50 starting hematopoietic precursors for both experiments. Colonies are shown as the mean ± SEM of triplicate plates per well.