Relative total RNA levels of G0/G1- and S/G2/M-phase HSC isolated from CY/G-CSF–treated mice, assessed by pyronine Y/Hoechst staining
| . | Tissue . | Pyronine Y–relative mean fluorescence intensity . | |
|---|---|---|---|
| G0/G1 . | S/G2/M . | ||
| Experiment 1 | Untreated BM | 1.00 | 1.55 |
| CY/G-CSF day + 5 BM | 1.65 | 2.59 | |
| CY/G-CSF day + 5 blood | 1.24 | 2.17 | |
| Experiment 2 | Untreated BM | 1.00 | 1.48 |
| CY/G-CSF day + 4 BM | 1.02 | 1.54 | |
| CY/G-CSF day + 4 blood | 1.50 | 2.16 | |
| CY/G-CSF day + 4 spleen | 1.38 | 2.01 | |
| Experiment 3 | Untreated BM | 1.00 | 1.68 |
| CY/G-CSF day + 3 BM | 1.49 | 2.03 | |
| CY/G-CSF day + 4 BM | 1.11 | 1.64 | |
| CY/G-CSF day + 4 blood | 1.13 | 1.98 | |
| CY/G-CSF day + 4 spleen | 1.37 | 2.10 | |
| . | Tissue . | Pyronine Y–relative mean fluorescence intensity . | |
|---|---|---|---|
| G0/G1 . | S/G2/M . | ||
| Experiment 1 | Untreated BM | 1.00 | 1.55 |
| CY/G-CSF day + 5 BM | 1.65 | 2.59 | |
| CY/G-CSF day + 5 blood | 1.24 | 2.17 | |
| Experiment 2 | Untreated BM | 1.00 | 1.48 |
| CY/G-CSF day + 4 BM | 1.02 | 1.54 | |
| CY/G-CSF day + 4 blood | 1.50 | 2.16 | |
| CY/G-CSF day + 4 spleen | 1.38 | 2.01 | |
| Experiment 3 | Untreated BM | 1.00 | 1.68 |
| CY/G-CSF day + 3 BM | 1.49 | 2.03 | |
| CY/G-CSF day + 4 BM | 1.11 | 1.64 | |
| CY/G-CSF day + 4 blood | 1.13 | 1.98 | |
| CY/G-CSF day + 4 spleen | 1.37 | 2.10 | |
Thy-1.1loSca-1+ Lin−c-Kit+ cells were analyzed by 8-parameter flow cytometry. Emitted fluorescence from Hoechst-33342 was detected in the UV channel, and fluorescence from pyronine Y was detected in the phycoerythrin (PE) channel. To enable comparison among the 3 experiments, mean fluorescence intensity for pyronine Y was normalized to the levels recorded for G0/G1 cells from bone marrow of untreated mice in each experiment. The boundary between G0/G1 and S/G2/M cells was established as shown in Figure 4, which is from experiment 1.