Nocodazole treatment of megakaryocytes.

Nocodazole (▪, 2 μg/mL) was added to the medium of culture of megakaryocytes obtained from CD34⁺ cells with PEG-rhuMGDF and SCF. Megakaryocytes were regularly sampled (at 0, 3, 5, 7, and 24 hours) centrifuged onto slides and fixed with paraformaldehyde, then nuclei were counterstained with DAPI. Mitotic index was calculated for 400 cells at each time and determined on the average of triplicate experiments. In parallel, nocodazole was withdrawn (▨) at 7 hours and mitotic indexes were similarly determined at 24 and 48 hours (A). Panel B shows a similar experiment in which only polyploid megakaryocytes were taken into account to determine the mitotic index (based on a 500-cell count for each sample).