The purification of murine CFU-MK
| Experiment No. . | Purification Step . | Recovery . | No. MK Colonies (per 105 cells) . | Fold Enrichment . | |
|---|---|---|---|---|---|
| Cell . | CFU-MK . | ||||
| 1 | Whole marrow | (100%) | (100%) | 42 ± 3 | 1 |
| Density gradient | 48% | 95% | 110 ± 20 | 2.6 | |
| Lineage depletion | 3.2% | 58% | 760 ± 114 | 18 | |
| FACS sorting | 0.03% | 8.3% | 11400 ± 2030 | 271 | |
| 2 | Whole marrow | (100%) | (100%) | 44 ± 11 | 1 |
| Density gradient | 51% | 100% | 92 ± 15 | 2.1 | |
| Lineage depletion | 3.3% | 63% | 847 ± 116 | 19.3 | |
| FACS product | 0.02% | 6.1% | 11700 ± 3770 | 265 | |
| Experiment No. . | Purification Step . | Recovery . | No. MK Colonies (per 105 cells) . | Fold Enrichment . | |
|---|---|---|---|---|---|
| Cell . | CFU-MK . | ||||
| 1 | Whole marrow | (100%) | (100%) | 42 ± 3 | 1 |
| Density gradient | 48% | 95% | 110 ± 20 | 2.6 | |
| Lineage depletion | 3.2% | 58% | 760 ± 114 | 18 | |
| FACS sorting | 0.03% | 8.3% | 11400 ± 2030 | 271 | |
| 2 | Whole marrow | (100%) | (100%) | 44 ± 11 | 1 |
| Density gradient | 51% | 100% | 92 ± 15 | 2.1 | |
| Lineage depletion | 3.3% | 63% | 847 ± 116 | 19.3 | |
| FACS product | 0.02% | 6.1% | 11700 ± 3770 | 265 | |
Low-density cells were separated from marrow cells by density gradient (density = 1.080), and lineage-committed cells were depleted with magnetic beads. Lineage-negative cells were then stained with FITC-conjugated CD41 and PE-conjugated c-kit monoclonal antibodies. CD41 positive and c-kit positive cells were sorted by FACStar plus. Cells were cultured in semisolid media with murine TPO (25 ng/mL), murine IL-3 (20 ng/mL), murine SCF (50 ng/mL), and human IL-6 (50 ng/mL) for 5 days. Whole marrow cells, low-density cells, lineage-negative cells, and sorted cells were plated at the concentration of 1 × 105, 1 × 104, 1 × 104, and 1 × 103/dish, respectively. The results represent the number of CFU-MK derived colonies (mean ± SEM of triplicate plates).