Determining the effect of red cell microvesicles isolated from stored blood on THP-1 cells
Cardiff Metropolitan University
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Purpose. The aim of the experiment was to establish whether stored whole blood and erythrocyte blood samples produce a pro or anti inflammatory effect as a consequence of interaction of erythrocyte derived microvesicles with monocytic THP-1 cells. ‘THP 1 cells only’ and ‘THP 1 cells with LPS’ acted as negative and positive controls and any change of cytokine production levels when different amounts of SAG-M and WB microvesicles were added (500, 1000 or 2000) would suggest dose dependent response. Methods. 2 different blood bags SAG-M (35 days old) and WB (2 days old) were centrifuged at 2 different speeds (17,000g and 100,000g). Running flow cytometer and NanoSight analysis, an approximation on how many microvesicles were found in each sample was made and using 4 x 24 well plates, either 2000, 1000 or 500 microvesicles were added to THP-1 cells and then exposed to 2 sandwich ELISA analysis: one for IL-1 Beta and one for IL-8, for both SAG-M and whole blood. Anova tests were carried in order to establish whether or not there was significant difference in proliferation of THP-1 cells and different level of IL-8 and IL-1 Beta production. Results. The results showed that microvesicles produced during a 35-day storage period of SAG-M, obtained through centrifugation at 17,000g produced a pro inflammatory effect without LPS stimulation, releasing both IL-8 and IL-1 Beta cytokines. It was also found that the addition of whole blood microvesicles has an immunosuppression effect on THP-1 cells regarding the production of IL-8. In conclusion, the effect of microvesicles produced by both SAG-M and whole blood during storage could have significant implications in blood transfusion, possibly causing or immunosuppressing an immune response, by the release of cytokines such as IL-8 and Il-1 Beta.
(BSc) Biomedical Science
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