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Abstract

Biology

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Published: August 7th, 2021

DOI:

10.3791/62569

1Platelet Research Lab, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), 2Flow Cytometry and Cell Sorting Platform (ISPA), 3Clinical Diagnosis Laboratory - Dept. of Hematology, Hospital Universitario Central de Asturias (HUCA), 4Department of Hematology, Hospital Universitario Severo Ochoa (HUSO), 5Department of Hematology, Instituto de Investigación Sanitaria San Carlos (IdISSC), Hospital Clínico San Carlos (HCSC), 6Dept. of Medicine, University of Oviedo

Abstract

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even >64N) and extremely large cell (40-60 µm). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

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Keywords Immunophenotyping

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