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    Using TIRF microscopy to analyze stimulated and basal state B-cell MHC II clustering in response to ageing and dietary fish oil

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    Author
    Melton, Mark T.
    Abstract
    This research focused on developing an efficient TIRF microscopy approach to evaluate membrane protein organization. More specifically, the data demonstrate that TIRF microscopy can detect changes in ex vivo B-cell MHC II lateral organization using a monoclonal antibody under differing conditions. MHC II clustering is dependent on the underlying lipid environment and upon LPS stimulation MHC II expression is dramatically increased. Using mice fed a fish oil or control diet for three weeks, or using mice aged for nine months, we imaged splenic B-cell MHC II clustering using TIRF microscopy. We also used LPS to stimulate B-cells from both experimental conditions to determine if either ageing the animals or feeding them fish oil could affect MHC II clustering. We then determined cluster quantity, size, and intensity using the NIH ImageJ software. The data showed that neither a relevant dose of fish oil, nor aging the mice approximately nine months, had an affect on MHC II clustering in the absence of LPS stimulation. However upon LPS stimulation, MHC II clustering dramatically changed in aged mice as well as fish oil fed mice compared to control animals. Taken together, the data establish the TIRF microscopy protocol as a relevant alternative to more costly and time consuming approaches to address membrane protein clustering. Moreover, either ageing the animals or feeding them fish oil significantly affects MHC II clustering upon stimulation with LPS.  
    URI
    http://hdl.handle.net/10342/4247
    Subject
     Chemistry, Biochemistry; Immunology; B cells; Lipid rafts; MHC class II; Plasma membrane; Protein clustering; TIRF; Biochemistry 
    Date
    2013
    Citation:
    APA:
    Melton, Mark T.. (January 2013). Using TIRF microscopy to analyze stimulated and basal state B-cell MHC II clustering in response to ageing and dietary fish oil (Master's Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/4247.)

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    MLA:
    Melton, Mark T.. Using TIRF microscopy to analyze stimulated and basal state B-cell MHC II clustering in response to ageing and dietary fish oil. Master's Thesis. East Carolina University, January 2013. The Scholarship. http://hdl.handle.net/10342/4247. October 01, 2023.
    Chicago:
    Melton, Mark T., “Using TIRF microscopy to analyze stimulated and basal state B-cell MHC II clustering in response to ageing and dietary fish oil” (Master's Thesis., East Carolina University, January 2013).
    AMA:
    Melton, Mark T.. Using TIRF microscopy to analyze stimulated and basal state B-cell MHC II clustering in response to ageing and dietary fish oil [Master's Thesis]. Greenville, NC: East Carolina University; January 2013.
    Collections
    • Biomedical Sciences
    • Master's Theses
    Publisher
    East Carolina University

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