Mature neutrophils account for the majority of hematopoietic cells in the ES/OP9 coculture system
. | Flushed wells* . | . | Flushed and trypsinized wells† . | . | ||
|---|---|---|---|---|---|---|
| Cell type . | Average no.‡ . | % recovered . | Average no.‡ . | % recovered . | ||
| Mature neutrophils | 500 000 | 79.7% ± 4.6% | 617 500 | 34.4%§ ± 7.5% | ||
| Monocytes/macrophages | 75 000 | 12%§∥ | 80 000 | 4.4% ± .35% | ||
| Erythroid | 50 000 | 8%§ | 254 556 | 14% ± .5% | ||
| Stroma | 2 500 | .4% | 850 000 | 47.2% ± .3% | ||
. | Flushed wells* . | . | Flushed and trypsinized wells† . | . | ||
|---|---|---|---|---|---|---|
| Cell type . | Average no.‡ . | % recovered . | Average no.‡ . | % recovered . | ||
| Mature neutrophils | 500 000 | 79.7% ± 4.6% | 617 500 | 34.4%§ ± 7.5% | ||
| Monocytes/macrophages | 75 000 | 12%§∥ | 80 000 | 4.4% ± .35% | ||
| Erythroid | 50 000 | 8%§ | 254 556 | 14% ± .5% | ||
| Stroma | 2 500 | .4% | 850 000 | 47.2% ± .3% | ||
As described in “Materials and methods,” wells from cultures from day 9 in the neutrophil differentiation mix were thoroughly flushed or flushed and trypsinized to include stromal cells, and the number and cell type evaluated using FACS and morphology as indicated. For FACS analysis, neutrophils, monocytes/macrophages, and erythroid cells were identified using antineutrophil Ag and Gr-1, CD-14, and Ter119 antibodies, respectively. For identification of stromal cells using FACS; CFDA SE staining of the OP9 cells was done before hematopoietic cells were plated onto them at the secondary and tertiary differentiation stages. Data are shown ± standard deviations.
Cells from a single flushed well were recovered from gentle and thorough harvesting.
Cells from a single trypsinized well were recovered following gentle and thorough harvesting.
Based on hemacytometer counting.
Based on morphology.
Based on morphology. Between 70% to 85% of these cells are mature neutrophils, while monocytes/macrophages generally account for approximately 12% to 20% of the nontrypsinized cells at day 9 in the neutrophil differentiation mix.