Surface Ultrastructure of Human Megakaryocytes Sorted on the Basis of DNA Content

The relationship of polyploidization (DNA content) to differentiation is not well defined. We have developed centrifugal elutriation and Percoll density gradient centrifugation to obtain larye numbers of highly-purified mega-karyocytes which subsequently were stained for DNA content with Hoechst 33342 and sorted by FACS into SC, 16C and 32C ploidy classes for correlated analysis of cell surface structures by scanning electron microscopy. Each ploidy class revealed unique surface characteristics that reflect differentiation occurring in megakaryocytes independent of their DNA content

Separation of megakaryocytes from mouse bone marrow by density gradient centrifugation

Abstract The density profile of mouse bone marrow megakaryocytes, as determined by discontinuous albumin density gradient centrifugation, was characterized by a single density population (1.088 to 1.119 g/ml) with a peak density of 1.10 g/ml and a maximum enrichment of 15.5. This single population comprised both mature and immature megakaryocytes. The density profile of small, acetylcholinesterase-positive cells, a class of potential megakaryocyte precursors, was almost identical to that of morphologically recognizable megakaryocytes.</jats:p

Separation of Megakaryocytes From Mouse Bone Marrow by Velocity Sedimentation

Abstract A technique for separating mouse bone marrow megakaryocytes by unit gravity velocity sedimentation is described using a modified sedimentation chamber. The velocity sedimentation profile for megakaryocytes comprises three peaks corresponding to sedimentation velocities of 30, 60, and 100 mm/hr. Morphologic examination of megakaryocytes in the 30 mm/hr peak reveals them to be primarily immature with the latter two peaks corresponding to larger, more mature megakaryocytes.</jats:p

Thromboxane synthesis in megakaryocytes isolated by centrifugal elutriation

A systematic approach to the optimization of centrifugal elutriation is described for the purification of rat femoral marrow megakaryocytes, which has permitted the first direct demonstration of thromboxane synthesis in these cells. The rapidity, simplicity, and capacity of this technique has made it possible to process 2 x 10(9) marrow cells in the absence of platelet aggregation inhibitors in 20 min to obtain 10(6) megakaryocytes at a recovery of about 80% with a concentration of up to 282-fold. Incubation of these isolated megakaryocytes with 14C- arachidonic acid and subsequent thin-layer radiochromatography has revealed thromboxane B2 as the major cyclooxygenase pathway metabolite of arachidonic acid in megakaryocytes.</jats:p