Measuring Antigen-Specific T Cell Cytokines

The preparation of single-cell spleens was carried out as previously described [25 (link)]. 7 days after the boost immunization, splenocytes were stimulated with S. Typhimurium LPS (50 ng/µL) for 8 h at 37 °C with 5% CO₂ in the presence of a protein transport inhibitor mixture (Thermo Fisher). Cytokine expression of CD4⁺ T cells was measured using rat anti-mouse CD3-Alexa Fluor 700, CD4-FITC, IFNγ-APC/Cy7, and IL-4-Alexa Fluor 647 (eBioscience). The ratio of cytokine-positive cells in the unstimulated sample was subtracted from the antigen-stimulated value of the same mouse to calculate the percentage of antigen-specific cells.

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Han Y., Luo P., Zeng H., Wang P., Xu J., Chen P., Chen X., Chen Y., Cao Q., Zhai R., Xia J., Deng S., Cheng A., Cheng C, & Song H. (2023). The effect of O-antigen length determinant wzz on the immunogenicity of Salmonella Typhimurium for Escherichia coli O2 O-polysaccharides delivery. Veterinary Research, 54, 15.

Publication 2023

protein transport inhibitor mixture CD3-Alexa Fluor 700 CD4-FITC IFNγ-APC/Cy7

A protein Alexa fluor 647 Anti cd3 Antigen Cd4 t cells Cytokine Fitc Ifnγ Immunization Mouse Protein transport

Corresponding Organization :

Other organizations : Zhejiang A & F University, Sichuan Agricultural University

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Variable analysis

independent variables

Boost immunization

dependent variables

Cytokine expression of CD4+ T cells (IFNγ and IL-4)

control variables

Time point (7 days after boost immunization)
Stimulation with S. Typhimurium LPS (50 ng/µL) for 8 h at 37 °C with 5% CO2
Presence of protein transport inhibitor mixture

controls

Positive control: Stimulation with S. Typhimurium LPS
Negative control: Unstimulated sample

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