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A fluorescence microscopy method for quantifying the detection of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells

Biological Procedures Online volume 3, pages 54–63 (2001)Cite this article

Abstract

In platelets, PGHS-1-dependant formation of thromboxane A2 is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are anucleated cells, we have used the immortalized human megakaryoblastic cell line MEG-01, which can be induced to differentiate into platelet-like structures upon addition of TPA as a model system to study PGHS-1 gene expression. Using a specific antibody to PGHS-1 we have developed a technique using immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein expression as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 × 10−8 M) over a period of 8 days. The method represents a rapid and economical alternative to flow cytometry. Using this technique we observed that TPA induced adherence of MEG-01 cells, and only the non-adherent TPA-stimulated cells demonstrated compromised viability. The differentiation of MEG-01 cells was evaluated by the expression of the platelet-specific cell surface antigen, CD-41. The latter was expressed in MEG-01 cells at the later stages of differentiation. We demonstrated a good correlation between PGHS-1 expression and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein expression, which shows a consistent increase over the entire course of differentiation can be used as an additional and better index by which to monitor megakaryocyte differentiation.

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, K1H 8M5, Ottawa, Ontario, Canada

    Douglas J. Franks

  2. Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, K1H 8M5, Ottawa, Canada

    Cameron Mroske & Odette Laneuville

Authors
  1. Douglas J. Franks
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  2. Cameron Mroske
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  3. Odette Laneuville
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Correspondence to Douglas J. Franks.

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Published: December 12, 2001

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Franks, D.J., Mroske, C. & Laneuville, O. A fluorescence microscopy method for quantifying the detection of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells. Biol Proced Online 3, 54–63 (2001). https://doi.org/10.1251/bpo23

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  • DOI: https://doi.org/10.1251/bpo23

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Biological Procedures Online

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