मिलान विशेष टी कोशिकाओं की MHC tetramer आधारित संवर्धन: पेप्टाइड
Summary
MHC tetramers और चुंबकीय microbeads मिलान विशेष टी कोशिकाओं की कम आवृत्ति आबादी को अलग करने और उन्हें प्रवाह cytometry द्वारा विश्लेषण: इस प्रोटोकॉल पेप्टाइड के उपयोग का वर्णन करता है. इस विधि अंतर्जात से ब्याज की टी सेल की आबादी के प्रत्यक्ष अध्ययन के लिए सक्षम बनाता है<em> Vivo में</em> प्रयोगात्मक सिस्टम.
Abstract
A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.
Protocol
Representative Results
Discussion
pMHC टेट्रामर आधारित सेल संवर्धन विधि इस प्रोटोकॉल द्वारा प्रस्तुत अंतर्जात टी सेल repertoires से मिलान विशेष टी कोशिकाओं का अध्ययन करने के लिए एक शक्तिशाली उपकरण है. मिलान विशेष टी कोशिकाओं के उनके TCRs की क्षमत?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए इस प्रोटोकॉल के विकास में मदद के लिए आंद्रे हान और लॉरेंस येन तकनीकी सहायता के लिए और जेनकींस प्रयोगशाला के सदस्यों को धन्यवाद करना चाहते हैं.
Materials
| Reagent | Vendor | Catalog number |
| PE or APC conjugated pMHC tetramer (or multimer) | Made by investigator, obtained from the NIH tetramer core, or purchased from commercial sources | |
| Anti-PE conjugated magnetic microbeads | Miltenyi | 130-048-801 |
| Anti-APC conjugated magnetic microbeads | Miltenyi | 130-090-855 |
| LS magnetic columns | Miltenyi | 130-042-401 |
| MidiMACS or QuadroMACS magnet | Miltenyi | 130-042-302 or 130-090-976 |
| Cell counting beads | Life Technologies | PCB-100 |
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