Time course of spontaneous proplatelet formation by collected megakaryocytes. We observed the production of spontaneous proplatelet formation in the same manner as the proplatelet formation inhibition assay in the absence of antibodies. Megakaryocyte colonies were derived from peripheral blood CD34+ cells (1.8 ± 0.5 × 104 cells/mL) stimulated with 10 ng/mL TPO and 10 ng/mL SCF, and they were collected on the 9th day from culture dishes (343.0 ± 3.0 colonies). Then, collected megakaryocytes were aliquoted into the wells of a 96-well tissue culture plate (190.9 ± 11.2 cells per well) and were incubated. The number of megakaryocytes with proplatelet formation was counted after 5, 24, and 48 hours of incubation, respectively, using an inverted microscope. The %PPF calculation is described in detail in the Materials and Methods section. The time 0 corresponds with the 9th day of culture in Fig 3. The purity of the GP-IIb-positive cells was 98.9% ± 0.3% per well, by indirect immunostaining. The data are the means ± SEM from duplicate incubations of two experiments.
