CCE-25–c-Kit^negSca-1^pos and Sca-1^neg cell growth in “noncontact” M2-10B4 cocultures. Purified CCE-25–c-Kit^negSca-1^pos and Sca-1^neg cells were added to M2-10B4 monolayers and were cocultured in transwells over M2-10B4 monolayers for 25 days with biweekly feeding in the presence of 100 ng/mL SCF and 30 ng/mL IL-3. Cells were harvested and stained as described in “Materials and methods.” (A) Flow cytometry for myeloid lineage markers Gr-1 and Mac-1. (B) Flow cytometry for PHSC markers c-Kit and Sca-1. These data are representative of 3 points per group in 3 separate experiments. For these analyses, background staining, indicated by staining with isotype-matched controls, was subtracted to yield the percentages shown on the dot plots. (C) The morphology of CCE-25–c-Kit^negSca-1^pos and Sca-1^neg cells grown in “noncontact” M2-10B4 cultures over time. Photomicrographs of purified CCE-25–c-Kit^negSca-1^pos and Sca-1^neg cells that were cocultured in transwells over M2-10B4 monolayers for 25 days with biweekly feeding in the presence of 100 ng/mL SCF and 30 ng/mL IL-3. Cells were harvested and stained as described in “Materials and methods.” At 18 days after seeding, cells were harvested and cytospun. Mφ indicates macrophage morphology; mast, mast cell morphology.