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Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay
Published on: October 30, 2015
Quantitating MHC class I ligand production and presentation using TCR-like antibodies
1Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. dolanbr@mail.nih.gov.
Methods in Molecular Biology (Clifton, N.J.)
|January 19, 2013
Summary
Quantify peptide-MHC class I complexes on cell surfaces using flow cytometry. This method aids in understanding antigen presentation and T cell activation by counting specific complexes.
Area of Science:
- Immunology
- Cell Biology
Background:
- Accurate quantification of cell surface peptide-MHC class I complexes is crucial for assessing antigen presentation.
- Understanding these complexes is vital for evaluating cellular processes and pharmaceutical effects on the immune system.
Purpose of the Study:
- To present a quantitative flow cytometry method for determining peptide-MHC class I complex numbers on cultured cells.
- To enable precise measurement of specific peptide-MHC complexes for research applications.
Main Methods:
- Utilizes a monoclonal antibody with specificity for the target peptide-MHC class I complex.
- Employs fluorescently calibrated beads to quantify antibody binding and thus complex numbers.
- Applies flow cytometry to cells cultured in vitro expressing the relevant endogenous protein.
Main Results:
- The described method allows for the enumeration of specific peptide-MHC class I complexes on the cell surface.
- Provides a quantitative measure of antigen presentation at the cellular level.
Conclusions:
- This flow cytometry application offers a reliable way to quantify peptide-MHC class I complexes.
- The method supports research into T cell activation and the development of immunomodulatory pharmaceuticals.
