Lack of effect of low-dose tumor necrosis factor on the viability and clonal growth of individual CD34+CD38− human marrow cells assessed in single-cell cultures
| Period of culture (days) . | TNF addition . | Proportion (%) of wells containing different numbers of cells . | ||
|---|---|---|---|---|
| 0 cells . | 1 cell . | 2 cells . | ||
| 3 | − | 4.3 ± 1.1 | 82 ± 5 | 14 ± 5 |
| + | 7.8 ± 1.7 | 81 ± 3 | 12 ± 4 | |
| 10 | − | 27 ± 8 | 34 ± 15 | 40 ± 20 |
| + | 32 ± 7 | 32 ± 13 | 36 ± 18 | |
| Period of culture (days) . | TNF addition . | Proportion (%) of wells containing different numbers of cells . | ||
|---|---|---|---|---|
| 0 cells . | 1 cell . | 2 cells . | ||
| 3 | − | 4.3 ± 1.1 | 82 ± 5 | 14 ± 5 |
| + | 7.8 ± 1.7 | 81 ± 3 | 12 ± 4 | |
| 10 | − | 27 ± 8 | 34 ± 15 | 40 ± 20 |
| + | 32 ± 7 | 32 ± 13 | 36 ± 18 | |
TNF indicates tumor necrosis factor; FACS, fluorescence-activated cell sorting; FL, Flt3 ligand; SF, Steel factor; IL, interleukin; G-CSF, granulocyte colony-stimulating factor.
Annexin V− PI−CD34+CD38− cells were isolated from low-density normal human marrow samples and deposited directly using the FACS into the round-bottomed wells of 96-well plates preloaded with serum-free medium containing FL and SF (at 100 ng/mL) and IL-3, IL-6, and G-CSF (at 20 ng/mL), with or without 0.1 ng/mL TNF, as indicated. Cultures were assessed by visual examination at the times shown, and the proportions of wells containing 0, 1, or more than 1 viable (refractile) cell per well were identified. Results shown are the mean ± SEM of data obtained in 5 independent experiments (with different marrow samples), in each of which at least 96 single-cell cultures were evaluated.