Second dose of CY.

Administration of a second dose of CY, given on day +2 of the standard CY/G-CSF protocol (Figure 1A; top), resulted in the disappearance of nearly all phenotypically defined LT-HSC from the BM on day +4, and the failed appearance of LT-HSC in the blood and spleen at day +4. Panel A shows the fluorescence profile of Thy-1.1^loSca-1⁺–gated BM, blood, or spleen cells for lineage and c-Kit staining. The gates for LT-HSC (Lin⁻ c-Kit⁺) are boxed. Cells in panel A (top) were isolated from a representative control mouse receiving the standard CY/G-CSF regimen (Figure 1A; top). Cells in panel A (bottom) were from a representative mouse that also received a second dose of CY on day +2 of the protocol. Mice in both groups were killed on day +4. The units of all plots (both axes) are log₁₀fluorescence intensity. Panel B shows the estimated number of LT-HSC per mouse for control animals, and for those that received the additional dose of CY on day +2 (control, n = 3 mice; 2nd dose CY, n = 4 mice). The number of LT-HSC was calculated by multiplying the obtained frequencies by the number of nucleated cells in each tissue, assuming that, mice BM recovered from the 4 long bones represents 15% of the total BM, and blood volume in milliliters is 10% of the animal's weight in grams.53