IL-1α–induced MK differentiation and rupture-dependent platelet biogenesis in vitro. (A–C) Time-lapse images of cultured MKs. Liver cells were collected from fetal CAG-eGFP mice on embryonic day 13 and cultured with TPO. On day 7 of culture, the cells were washed and incubated with anti-CD41 and Hoechst 33342. MKs were identified in the cultures as multinucleate and staining positive for CD41. MKs were then treated with TPO, IL-1α, or TPO plus Fas ligand 1 h before the experiments. Proplatelet production was observed in the presence of TPO, whereas MK rupture was seen in the presence of IL-1α. (D and E) Quantification of MK dynamics (D) and released particle size (E). n = 20 low-power fields (D) and n = 20 particles (E). Some cells were cultured with Z-VAD (OMe)-FMK (100 µM) 1 d before the experiments. (F) Fetal liver cells were isolated and cultured with TPO for 6 d. Differentiated MKs were washed and then incubated for 1 d with TPO, IL-1α, and anti–IL-1α neutralizing antibody. Ploidy was analyzed in CD41⁺CD42b⁺Lin⁻ MKs. The data shown are from a single representative experiment from among three repeats. (G and H) BM cells isolated from 6-wk-old WT mice were cultured, and CD41⁺CD42b⁺Lin⁻ MKs were counted (G) in each well after 7 d. The percent value was normalized to that of a control well. n = 5 experiments. (H) Release of CD41⁺CD42b⁺ particles into the culture medium. n = 5 experiments. *, P < 0.05.