Analysis of hematopoietic reconstitution after transplantation of BM cells into lethally irradiated recipients.

(A) BM cells were harvested from 4- to 6-week-old STAT5ab^−/− mice (C57Bl/6 Hb_s). The cells were then injected into lethally irradiated (1100 rads) C57Bl/6 HW80 recipient mice (Hb_d) that differ in the endogenous hemoglobin expression pattern. Eight to 10 weeks later the mice were bled, and the packed red blood cells were separated by electrophoresis on cellulose acetate gels. The Hb_s and Hb_dpatterns of control mice that did not receive transplants are shown on the right. Shown are representative examples of mice that received transplants 5 (BMT no. 1), 6 (BMT no. 2), and 3 months (BMT no. 3) earlier of STAT5ab^−/− BM cells. Also shown are secondary BM transplanted mice from primary BM transplantation no. 1 analyzed 3 months after transplantation. (B) In a fourth experiment, BM cells (Ly-5.2) were injected into lethally irradiated Ly-5.1 mice, and then peripheral blood leukocytes were stained with antibodies and analyzed by FACS after reconstitution. Shown are representative examples of mice analyzed for Ly-5.2 expression 5 months after transplantation. Ly-5.1 negative control and Ly-5.2 positive controls are shown in the left panels. On the right are mice that received transplants of wild-type BM (upper panel) or STAT5ab^−/− BM cells (lower panel). (C) To demonstrate a typical lineage analysis of the STAT5ab^−/− BM engraftment, gating based on forward-scatter and side-scatter profiles typical for lymphocytes (B220, Thy1.2, and Ter119), monocytes (Mac-1), and granulocytes (Gr-1) was combined with the lineage antibodies indicated. The percentage of cells falling within the upper quadrants and the lower-right quadrant is shown. Note that for the Thy1.2 analysis cells in the upper-left quadrant were slightly overcompensated, making the percentage in the quadrant appear visually lower than the actual value.