Departmental Affiliation and City, State, Zip for All Authors

Corbin McElrath, Old Dominion University, Norfolk, Virginia, 23529; Dong-Ming Su, Department of Cell Biology, Immunology, and Microbiology, UNT Health Science Center, Fort Worth, Texas, 76107; Rachel Thomas, Department of Cell Biology, Immunology, and Microbiology, UNT Health Science Center, Fort Worth, Texas, 76107; Jiyoung Oh, Department of Cell Biology, Immunology, and Microbiology, UNT Health Science Center, Fort Worth, Texas, 76107

Scientific Abstract

Optimal staining conditions for the MHC class II tetramer vary from lab to lab as there is no definitive and established procedure used throughout the field of immunology. In this experiment, we attempted to test the efficacy of a reagent of MHC Class II tetramer from NIH tetramer core used in flow cytometry of our project. This reagent is supposed to be useful for determination of mechanistic thymic involution, negative selection, and the production of regulatory T cells. The MHC class II tetramer used presents the OVA 323-339 self-peptide to the OT-II T Cell Receptor. The FF mouse, which expresses the wild type phenotype, was used as a control and an OT-II/RagKO mouse was used to analyze the binding of the MHC Class II tetramer because the T cells of the OT-II/RagKO transgenic mouse recognize ovalbumin as a mock self antigen. The thymus and spleen were harvested and homogenized from each mouse. Cells were properly stained with the respective antibodies for flow cytometric analysis to identify if the conditions were optimal for staining. The results illustrated negligible binding differentiation between the isotype control and the tetramer, indicating that these conditions were not optimal for the identification of OT-II+ T cells.

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TROUBLESHOOTING OF OVA 323-339 TETRAMER STAINING FOR FLOW CYTOMETRY

Optimal staining conditions for the MHC class II tetramer vary from lab to lab as there is no definitive and established procedure used throughout the field of immunology. In this experiment, we attempted to test the efficacy of a reagent of MHC Class II tetramer from NIH tetramer core used in flow cytometry of our project. This reagent is supposed to be useful for determination of mechanistic thymic involution, negative selection, and the production of regulatory T cells. The MHC class II tetramer used presents the OVA 323-339 self-peptide to the OT-II T Cell Receptor. The FF mouse, which expresses the wild type phenotype, was used as a control and an OT-II/RagKO mouse was used to analyze the binding of the MHC Class II tetramer because the T cells of the OT-II/RagKO transgenic mouse recognize ovalbumin as a mock self antigen. The thymus and spleen were harvested and homogenized from each mouse. Cells were properly stained with the respective antibodies for flow cytometric analysis to identify if the conditions were optimal for staining. The results illustrated negligible binding differentiation between the isotype control and the tetramer, indicating that these conditions were not optimal for the identification of OT-II+ T cells.

Manuscript Number

1021