TRAIL-mediated induction of apoptosis in erythroid precursors at intermediate stages of differentiation.

Seven-day unilineage erythroid (SCF + IL-3 + EPO) (A and B) and megakaryocytic (IL-3 + TPO) (A) cultures were incubated with irrelevant IgM mAb and rHis6-tag peptide (CONT), TRAIL, or anti-CD95 (CH 11) IgM mAb for 20 hours. (A) Erythroid and megakaryocytic cells were phenotypically characterized by double staining with anti-CD71 + anti-glycophorin A or anti-CD41 + anti-CD42b, respectively (upper 2 panels). Percentages of glycophorin A⁺ and CD41⁺ cells are indicated in each panel. Apoptotic erythroid cells were detected by staining with annexin V combined to glycophorin A, whereas apoptotic megakaryocytes were detected by propidium iodide staining combined to CD61 (lower 6 panels). Percentages of cells in the respective quadrants are indicated. Quadrants were set based on negative controls stained with isotype-matched irrelevant mAbs (not shown). Panel (B) shows the morphological analysis of erythroblasts stained with May-Grunwald-Giemsa. Apoptotic cells are indicated by arrowheads. Original magnification 600×.