Simultaneous flow cytometric analysis of megakaryocyte polyploidy and a labile intracellular protein using zinc-based...

Differentiating megakaryocytes undergo a unique endomitotic cell cycle leading to large polyploidal cells, which fragment to generate platelets, blood cells important for normal hemostasis. Simultaneous assessment of DNA content and cellular proteins by flow cytometry is a useful tool to study megakaryocyte differentiation and to define expression of proteins important for megakaryocyte development and platelet formation. The usefulness of zinc salt-based fixation (ZBF), a non-crosslinking method of cell fixation that permits downstream analysis of nucleic acids (Jensen et al., Cytometry A 2010;77A:798-804), in flow cytometric analysis of megakaryocyte ploidy in conjunction with extracellular and intracellular proteins was assessed. ZBF of a megakaryocyte-like cell line resulted in preservation of proteins similar to paraformaldehyde fixation, and preservation of DNA content in a manner similar to methanol fixation. This is highlighted by experiments in which polyploidal megakaryocytes were analyzed simultaneously for endocytosis of a fluorescently-labeled, endocytosed labile protein or expression of a cell surface integrin and DNA content. These studies demonstrate that ZBF will be a valuable tool to study the molecular events leading to platelet formation. © 2017 International Society for Advancement of Cytometry.