| Autor: |
Laurie Landry, Sarah Danielson, Amanda Anderson, Maki Nakayama |
| Jazyk: |
angličtina |
| Rok vydání: |
2021 |
| Předmět: |
|
| Zdroj: |
Bio-Protocol, Vol 11, Iss 2 (2021) |
| Druh dokumentu: |
article |
| ISSN: |
2331-8325 |
| DOI: |
10.21769/BioProtoc.3883 |
| Popis: |
Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear factor of activated T-cells (NFAT) signaling and read by flow cytometry. Reporter T-cells also constitutively express additional pairs of fluorescent proteins as identifiers, allowing for multiplexing of up to eight different reporter T-cell lines simultaneously, each expressing a different TCR of interest and distinguishable by flow cytometry. Once TCR expression cell lines are made they can be used indefinitely for making new T-cell lines with just one transduction step. This multiplexing system permits screening numbers of TCR-antigen interactions that would otherwise be impractical, can be used in a variety of contexts (i.e., screening individual antigens or antigen pools), and can be applied to study any T-cell-MHC-antigen trimolecular interaction. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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