SP cells are homogeneous for CD34 expression. (A) Nonspecific staining at high concentrations of RAM34 antibody. Wild-type (WT) and CD34-null BM cells were stained with Hoechst and subsequently stained with standard concentrations (0.2 μg) of anti-lineage, Sca-1, c-kit, and the indicated concentrations of RAM34-FITC. Histograms shown are gated on KLS-SP cells. Numbers within each panel represent the mean fluorescent intensity (MFI) for the FITC channel. (B) No difference in CD34 mRNA expression within SP cells. Whole bone marrow from wild-type mice (top left) was stained with Hoechst and antibodies against c-kit, Sca-1 lineage markers, and RAM34-FITC at 2.5μg/10⁶ cells. SP cells (gated as in left panel) that were lineage^–, c-kit⁺, and Sca-1⁺ were displayed for CD34 and sorted with the gates shown (top right). (C) Representative reanalysis of sorted CD34^– and CD34⁺ cells. (D) CD34 mRNA levels of sorted populations were analyzed by real-time PCR. Error bars represent SD. P < .5 (t test).