Proplatelet assay.

Bone marrow cells from BDF1 mice were cultured in TPO-containing media for 72 hours. Mature megakaryocytes (MKs) were purified by unit gravity sedimentation and then cultured in the presence of 10% human plasma for 48 additional hours with or without pharmacologic inhibitors. All experimental conditions contained 50 ng/mL recombinant human TPO. The number of proplatelet-bearing MKs was then enumerated by visual inspection of undisturbed cells. MTT was then added to the culture to reflect the total number of living cells. The data shown are representative results from 3 separate experiments. In each experiment, proplatelet-bearing MK counts were performed in 4 to 5 separate wells per condition tested. (A) A proplatelet-bearing MK. (B) The effects of PKC inhibitors on proplatelet formation. Bis-indolylmaleimide I (500 nmol/L) and prolonged incubation of PMA (100 ng/mL) decreased the number of proplatelet-forming MKs. The numbers of viable MKs measured by MTT assay (shown in the right set of bars) are only modestly affected. ░, DMSO; ▧, BIM 0.5 μM; ▨, PMA 100 ng/mL.