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

摘要

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.