Flow cytometry analysis of the effect of rapamycin on the cell cycle of megakaryocytes. CD34⁺ cells were cultured in the presence of TPO and Rapa (100 nM; TPO + Rapa) or without Rapa (TPO). (A) Apoptosis in cycling or resting MKs was evaluated by staining with Annexin V and 7AAD at day 6 of culture. (B) Analysis by BrDU incorporation and propidium iodide (PI) staining of G₁, S, and G₂/M phases of cell cycle at day 3 (D3) or day 6 (D6) in culture. (C) Rapa was added at days 0, 3, and 6 to the culture. Only CD42⁺ MKs were analyzed for ploidy level by PI staining at day 9 of culture. The mean ploidy was calculated from the number of cells of each ploidy level. (D) Rapa was added at day 3 and day 6 to the culture. The analysis of ploidy level was performed as in panel C. Each figure shows 1 representative analysis. Two additional experiments were performed for panels A and B and 3 for panels C and D with similar results.