Purification of human megakaryocytes by fluorescence-activated cell sorting

摘要: For direct studies of growth control, a method was developed to purify viable human megakaryocytes homogeneity from routine normal bone marrow aspirates. An initial separation over 1.050 g/mL Percoll density cut used enrich megakaryocytes. After washing, the cells were specifically labeled with fluoresceinated monoclonal antibody or F(ab9)2 fragment platelet glycoprotein (GP) IIb/IIIa complex. Megakaryocytes selectively sorted by using Becton Dickinson FACStar flow cytometer on basis fluorescence intensity greater than 50-fold that control cells. To increase resolution and purity sorting rate adjusted one cell in 13 formed drops, negative events coincided positive ones aborted. Two thirds isolated large, morphologically recognizable forward light scatter fourfold main population. Microscopic examination showed these be equal 98% diameter 20 46 microns ploidy range 2N 64N mode 16N. The small highly fluorescent 10 21 diameter, their 32N classes 4N. majority also positively reacted GPIb. cultured either Iscove9s leucine, lysine-deficient RPMI 1640 medium 10% plasma. maintained culture more three days capable synthesis both DNA protein as assessed radiolabeled thymidine amino acid incorporation. Moreover, responding recombinant granulocyte-macrophage colony- stimulating factor. data show can purified aspirates lineage marker they are vitro.