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Related Experiment Video

Updated: Jun 6, 2026

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling
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Published on: October 28, 2014

Single-Cell Translation and Apoptosis Profiling to Define Human CD34 + Cell Response to Specific Factors.

Dan Li, Karin Gustafsson, Jelena Milosevic

    Biorxiv : the Preprint Server for Biology
    |June 5, 2026
    PubMed
    Summary

    This study introduces a novel flow cytometry method to measure protein synthesis and apoptosis in hematopoietic stem cells (HSCs). The assay helps understand how compounds like UM729, SR1, and dmPGE2 affect HSC function, aiding research into myelodysplastic syndromes.

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    Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

    Published on: November 11, 2014

    Area of Science:

    • Hematology
    • Cell Biology
    • Biochemistry

    Background:

    • Global mRNA translation is crucial for hematopoietic stem cells (HSCs) and often dysregulated in myelodysplastic syndromes (MDS).
    • Measuring protein synthesis and apoptosis simultaneously at single-cell resolution in HSC subpopulations is technically difficult.
    • Existing methods lack the capacity for parallel assessment of these critical cellular processes in defined HSC subsets.

    Purpose of the Study:

    • To develop and validate a single-tube flow cytometry protocol for simultaneous quantification of global protein synthesis and apoptosis in human cord blood (CB) CD34+ cells.
    • To assess the impact of specific small molecules (SR1, UM729, dmPGE2) on HSC function, including protein synthesis, apoptosis, and cell cycling.
    • To establish a transferable analytic platform for evaluating functional states of patient-derived HSCs, particularly in myeloid neoplasms associated with translational dysregulation.

    Main Methods:

    • Developed a single-tube flow cytometry protocol using O-propargyl-puromycin (OP-Puro) for global protein synthesis measurement and cleaved Caspase-3 for apoptosis detection.
    • Applied immunophenotyping to analyze canonical CD34+ HSC hierarchy in cryopreserved human cord blood (CB) CD34+ cells.
    • Conducted a four-condition factor-omission analysis of an ex vivo expansion cocktail (SR1 + UM729 + dmPGE2) across three independent CB donors.

    Main Results:

    • The protocol successfully quantifies protein synthesis and apoptosis in defined primary hematopoietic cell subsets using standard flow cytometry.
    • UM729 was found to constrain protein synthesis and promote apoptosis across the HSC hierarchy.
    • SR1 maintained a pro-survival state without affecting translation, while dmPGE2 promoted HSC cycling and exit from the primitive state.

    Conclusions:

    • The developed flow cytometry assay provides a robust method for assessing dynamic cellular processes in primary hematopoietic cells.
    • The study resolved distinct functional signatures for UM729, SR1, and dmPGE2, elucidating their mechanistic effects on HSCs.
    • This platform is proposed as a valuable tool for analyzing functional states in patient-derived CD34+ cells from MDS and other myeloid neoplasms.