Treatment protocols.

(A) CY/G-CSF dosing. Mice were injected intraperitoneally (IP) with a single dose of CY (4 mg) and injected subcutaneously (SC) on successive days with recombinant human G-CSF (5 μg) as shown. In one experiment, mice were also injected with a second dose of CY (4 mg) on day +2 (dashed arrow). (B) BrdU protocol. Mice were injected with CY and G-CSF as in panel A. In addition, they were given 4 mg BrdU as an intraperitoneal injection on day −1, and placed on BrdU-containing water for the duration of treatment. In one experiment (Table 1, experiment 1), mice also received daily intraperitoneal injections of BrdU on days 0, +1, +2, and +3 as shown (dashed arrows). These daily intraperitoneal injections were subsequently found not to increase BrdU labeling, and discontinued. (C) Flow cytometry plots of CY/G-CSF day +4 bone marrow (i, i′), and blood (ii, ii′) nucleated cells. Plots i and i′ and plots ii and ii′ are of the same BM and blood samples, respectively. Plots i′ and ii′ show the Sca-1 and c-Kit staining profiles of the Thy-1.1^loLin⁻ cells that are boxed in i and ii, respectively. Boxes represent FACS gates used to isolate LT-HSC. All plots show cells after gating out of dead cells. The units of all plots (both axes) are log₁₀ fluorescence intensity. (D) Representative photomicrographs of control thymocytes and of LT-HSC from BrdU-treated mice. In each case, the left and right panels depict the same field, viewed either through a UV filter for visualization of Hoechst 33342 staining of all nuclei (blue staining), or a FITC/Texas Red filter for visualization of anti-BrdU antibodies (green staining), respectively. Thymocytes from untreated mice (i, i′), or BrdU-treated mice (ii, ii′), were double sorted and stained with anti-BrdU antibodies. Thy-1.1^loSca-1⁺Lin⁻c-Kit⁺Mac-1⁻cells were isolated from BM (iii, iii′) and peripheral blood (iv, iv′) after treatment of mice with BrdU and CY/G-CSF (day +4) (original magnification × 125).