TNF indicates tumor necrosis factor; CFC, colony-forming cell; LTC-IC, long-term culture-initiating cell; FACS, fluorescence-activated cell sorting; FL, Flt3 ligand; SF, Steel factor; IL, interleukin; G-CSF, granulocyte colony-stimulating factor.

FACS-purified suspensions of Annexin V⁻ PI⁻CD34⁺CD38⁻ cells were isolated from the light-density fraction of normal human marrow samples, and 1200 of these cells per treatment group (ie, 6 × 100 μL cultures each containing 200 cells) were incubated for 7 days in serum-free medium supplemented with FL and SF (at 100 ng/mL each) and IL-3, IL-6, and G-CSF (at 20 ng/mL each). At the end of the first 2 days, the entire culture was restained with FITC-Annexin V, and any new Annexin V⁺ cells (3% in this case) were removed by resorting the cells. The Annexin V⁻ fraction was then resuspended in fresh medium containing the same 5 growth factors, with or without 0.1 ng/mL TNF. At the end of another 5 days at 37°C, the cells in each type of culture were harvested, pooled, and washed once (or not, as shown), and aliquots were assayed for CFCs and LTC-ICs. The numbers of directly detected CFCs and of CFCs produced by 6-week LTC-ICs in the serum-free suspension cultures at the end of the 5-day exposure to TNF are shown in comparison with the corresponding input values (or the postreselection values measured after the first 2 days in the absence of TNF), as indicated. Results are from 1 of 2 such experiments; similar findings were obtained in each.