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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dc.contributor.advisorShaikh, Saame Razaen_US
dc.contributor.authorMelton, Mark T.en_US
dc.contributor.departmentBiomedical Sciencesen_US
dc.date.accessioned2013-08-24T18:31:47Z
dc.date.available2014-10-01T14:45:52Z
dc.date.issued2013en_US
dc.description.abstractThis 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.  en_US
dc.description.degreeM.S.en_US
dc.format.extent48 p.en_US
dc.format.mediumdissertations, academicen_US
dc.identifier.urihttp://hdl.handle.net/10342/4247
dc.language.isoen_US
dc.publisherEast Carolina Universityen_US
dc.subjectChemistry, Biochemistryen_US
dc.subjectImmunologyen_US
dc.subjectB cellsen_US
dc.subjectLipid raftsen_US
dc.subjectMHC class IIen_US
dc.subjectPlasma membraneen_US
dc.subjectProtein clusteringen_US
dc.subjectTIRFen_US
dc.subjectBiochemistry
dc.subject.meshMicroscopy--methods
dc.subject.meshMembrane Microdomains
dc.subject.meshCell Membrane Structures
dc.subject.meshBiochemical Phenomena
dc.titleUsing TIRF microscopy to analyze stimulated and basal state B-cell MHC II clustering in response to ageing and dietary fish oilen_US
dc.typeMaster's Thesisen_US

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