The effect of gram negative lipopolysaccaride on CD14 expression in mononuclear white blood cells
Cardiff Metropolitan University
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Lipopolysaccaride (LPS) is one of the main components of the Gram negative cell wall, it gets released into the blood stream when bacterial cell are destroyed or lysed by the immune system. If the levels are allowed to build up it can cause sepsis, which is potentially life threatening. CD14 is a cell surface protein which is capable of binding LPS and setting off a series of reaction to neutralise it. In this experiment three types of cells, THP-1 Monocytes, differentiated THP Macrophages and MM6 Monocytes, where incubated with 5 different strain of LPS, there were also two incubation periods. The levels of membrane bound CD14 were measured by binding with monoclonal antibodies conjugated with a fluorochrome. The samples where then read by a flow cytometer in order to determine the change in CD14 levels from no LPS to incubated samples. The soluble CD14 was measured by ELISA and the results were read by a plate reader and then analysed in comparison to known standards in order to determine the exact levels. It was found that with Macrophages after 4 hours the levels of soluble CD14 rose greatly and the levels of membrane bound CD14 greatly fell possibly due to cleavage, however the overnight incubations revealed that the levels of membrane bound CD14 had fallen even further and the levels of soluble CD14 had also dropped below the original levels. With the Monocytes it was seen that after the 4 hour incubation the levels of hoth soluble and membrane bound CDl4 had fallen slightly, however after the overnight incubation they had greatly increased in some cases by 200% of the original value. When the results obtained where put through an ANOVA plot the P-value was revealed to be 0.001846 which signifies that the data is of a high statistical significance. The data found also concurs with several earlier studies, and conflicts with one.
BSc (Hons) Biomedical Science
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