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A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells
Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for...
में बचाया:
| मुख्य लेखकों: | , , , , , , , , , , , , |
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| स्वरूप: | लेख |
| भाषा: | अंग्रेज़ी |
| प्रकाशित: |
Philipps-Universität Marburg
2024
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| विषय: | |
| ऑनलाइन पहुंच: | पीडीएफ पूर्ण पाठ |
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| सारांश: | Understanding antigen-specific T-cell responses, for example, following virus
infections or allergen exposure, is of high relevance for the development of
vaccines and therapeutics. We aimed on optimizing immunophenotyping of T
cells after antigen stimulation by improving staining procedures for flow and
mass cytometry. Our method can be used for primary cells of both mouse and
human origin for the detection of low-frequency T-cell response using a dualbarcoding
system for individual samples and conditions. First, live-cell barcoding
was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation
assay. Second, to discriminate between stimulation conditions and prevent cell
loss, sample barcoding was combined with a commercial barcoding solution.
This dual-barcoding approach is cell sparing and, therefore, particularly relevant
for samples with low cell numbers. To further reduce cell loss and to increase
debarcoding efficiency of multiplexed samples, we combined our dualbarcoding
approach with a new centrifugation-free washing system by laminar
flow (Curiox™). Finally, to demonstrate the benefits of our established protocol,
we assayed virus-specific T-cell response in SARS-CoV-2–vaccinated and SARSCoV-
2–infected patients and compared with healthy non-exposed individuals by
a high-parameter CyTOF analysis. We could reveal a heterogeneity of
phenotypes among responding CD4, CD8, and gd-T cells following antigenspecific
stimulations. Our protocol allows to assay antigen-specific responses of
minute populations of T cells to virus-derived peptides, allergens, or other
antigens from the same donor sample, in order to investigate qualitative and
quantitative differences. |
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| वस्तु वर्णन: | Gefördert durch den Open-Access-Publikationsfonds der UB Marburg. |
| डिजिटल ऑब्जेक्ट पहचानकर्ता: | 10.3389/fcimb.2023.1336489 |