Intracellular staining for BDDpfVIII in bone marrow and spleen after transplantation with BDDpfVIII-transduced sca-1+ cells. Four mice were killed from both the 35 mg/kg BU + ATS, 3 × 105 cells and the 20 mg/kg BU + ATS, 106 cell groups. Bone marrow and spleen cells were harvested and analyzed by flow cytometry for intracellular BDDpfVIII staining, as well as surface markers for lineage differentiation. Representative data are shown for bone marrow (A-D,L) and spleen (E-H) cells from mice that underwent transplantation. Negative controls are blood from a C57BL/6 mouse (I-K) and an isotype control (L) using a biotinylated mouse anti-hfVIII antibody that is not cross-reactive to porcine or murine fVIII. Panels A-H and L are gated on CD45+ donor cells. The mouse shown was engrafted with 34% and 47% GFP+ donor cells in the bone marrow and spleen, respectively.
