A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells
<p>In platelets, PGHS-1-dependant formation of thromboxane A<sub>2</sub> is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are a-nucleated cells, we have used the immortalized human megakaryob...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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BMC
2001-01-01
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| Series: | Biological Procedures Online |
| Subjects: | |
| Online Access: | http://www.biologicalprocedures.com/bpo/arts/1/23/m23.htm |
| _version_ | 1811287237023760384 |
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| author | Franks Douglas Mroske Cameron Laneuville Odette |
| author_facet | Franks Douglas Mroske Cameron Laneuville Odette |
| author_sort | Franks Douglas |
| collection | DOAJ |
| description | <p>In platelets, PGHS-1-dependant formation of thromboxane A<sub>2</sub> is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are a-nucleated 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 utilizing immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein levels as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 x 10<sup>-8</sup> 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 levels and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein level, 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. |
| first_indexed | 2024-04-13T03:13:58Z |
| format | Article |
| id | doaj.art-4db0dd8a6bb94d5aacd791dc0e5f9968 |
| institution | Directory Open Access Journal |
| issn | 1480-9222 |
| language | English |
| last_indexed | 2024-04-13T03:13:58Z |
| publishDate | 2001-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Biological Procedures Online |
| spelling | doaj.art-4db0dd8a6bb94d5aacd791dc0e5f99682022-12-22T03:04:57ZengBMCBiological Procedures Online1480-92222001-01-0131546310.1251/bpo23A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cellsFranks DouglasMroske CameronLaneuville Odette<p>In platelets, PGHS-1-dependant formation of thromboxane A<sub>2</sub> is an important modulator of platelet function and a target for pharmacological inhibition of platelet function by aspirin. Since platelets are a-nucleated 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 utilizing immunofluorescence microscopy and analysis of multiple digital images to monitor PGHS-1 protein levels as MEG-01 cells were induced to differentiate by a single addition of TPA (1.6 x 10<sup>-8</sup> 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 levels and the overall level of cellular differentiation of MEG-01 cells. Furthermore, PGHS-1 protein level, 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.http://www.biologicalprocedures.com/bpo/arts/1/23/m23.htmImmunocytochemistryFluorescence microscopyCell differentiation |
| spellingShingle | Franks Douglas Mroske Cameron Laneuville Odette A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells Biological Procedures Online Immunocytochemistry Fluorescence microscopy Cell differentiation |
| title | A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells |
| title_full | A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells |
| title_fullStr | A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells |
| title_full_unstemmed | A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells |
| title_short | A fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide H synthase-1 and CD-41 in MEG-01 cells |
| title_sort | fluorescence microscopy method for quantifying levels of prostaglandin endoperoxide h synthase 1 and cd 41 in meg 01 cells |
| topic | Immunocytochemistry Fluorescence microscopy Cell differentiation |
| url | http://www.biologicalprocedures.com/bpo/arts/1/23/m23.htm |
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