Phenotype of baboon CD34⁺ bone marrow cells after 10 days in allogeneic stromal and PMVEC coculture. Immunomagnetically selected CD34⁺ baboon marrow cells (A) were labeled with the lipophilic dye PKH-26 and placed onto preestablished allogeneic baboon marrow stromal cell (B and C) or PMVEC (D and E) monolayers as described in Fig 2. After 10 days of culture in the presence of exogenous human SCF, IL-3, IL-6, and GM-CSF, the cells were harvested and stained with the MoAbs K6.1 and OKT-10, which recognize the baboon CD34 and CD38 antigens, respectively, and secondarily labeled with APC (K6.1) and FITC (OKT-10). After first gating on forward and side scatter and propidium iodide exclusion to consider only live, nucleated cells, events positive for K6.1 (R3, top row) were gated for OKT-10 versus PKH-26 analysis (bottom row). The horizontal cursors define the PKH fluorescence of the freshly isolated and labeled cells; events subsequently below this line have undergone cell division. Machine settings were calibrated using reference microbeads to assure consistency of PKH-26 measurement. The vertical cursors delineate positive K6.1-APC (B and D) and OKT-10-FITC (C and E) fluorescence based on both negative controls consisting of isotype-matched irrelevant antibody secondarily labeled with APC or FITC and positive fluorescence of reference beads; events to the right of these cursors are positive. The data shown represent one of three baboon marrows tested.