CD34⁺-derived DCs express high levels of mRNA for Eph receptors. (Left) RT-PCR analysis of Eph expression in hematopoietic cells. cDNA was prepared from unactivated and PMA-ionomycin-activated freshly isolated PBMCs, blood T cells, and monocytes, tonsil B cells, or granulocytes generated in vitro. Monocytes, granulocytes, and B cells were resting or activated with PMA-ionomycin for 1 hour and 6 hours and pooled. T cells and PBMCs were activated with PMA-ionomycin for 6 hours. (Right) RT-PCR analysis of Eph expression in CD34⁺-derived DCs and monocyte-derived DCs generated in vitro. cDNA was prepared from cord blood CD34⁺ progenitors cultured in the presence of GM-CSF and TNF-α for 6 and 12 days, and for an additional 4 days with CD40L L cells, from monocytes cultured in the presence of GM-CSF and IL-4 for 6 days (unactivated), after 24-hour activation with CD40L L cells, or as a control from CD40L L cells. RT-PCR was carried out under standard conditions using 50 ng cDNA for 35 cycles. All cDNA samples were normalized according to the results of β-actin PCR amplification of 21, 28, and 35 cycles (only the amplification of 28 cycles is shown). The absence of genomic contamination was controlled in all RNA samples (before the reverse transcription step) by PCR amplification of β-actin with primers designed to amplify genomic DNA, as shown in the lower panel. Results are representative of 3 independent RT-PCR samples.