Intra-cytoplasmic Cytokine Staining (ICS): Optimizing antigen stimulation for measuring M. tuberculosis-specific T cell response
| Autor: | W. J. Britton, H. Muflihah |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
medicine.diagnostic_test
biology medicine.medical_treatment Stimulation Brefeldin A biology.organism_classification Molecular biology Peripheral blood mononuclear cell Flow cytometry Mycobacterium tuberculosis chemistry.chemical_compound Cytokine chemistry medicine Cytokine secretion Tumor necrosis factor alpha |
| Zdroj: | Medical Technology and Environmental Health ISBN: 9781003016700 |
| DOI: | 10.1201/9781003016700-51 |
| Popis: | Protective immunity induced by new tuberculosis (TB) vaccines was assessed by an immunology assay measuring cytokines. Intra-cytoplasmic cytokine staining (ICS) requires optimization as an easy-to use package kit is not available. This preliminary study aims to optimize the period of Mycobacterium tuberculosis (Mtb) antigen stimulation and inhibition of cytokine secretion. Three designs on the stimulation of peripheral blood mononuclear cells (PBMC) with Mtb culture filtrate protein (CFP) and additional protein transport inhibitor Brefeldin A were compared. The incubation period is 1 day or 5 days of CFP stimulation with Brefeldin A at the last 4 or 18 hours. The production of Th1 cytokines was analysed by flow cytometry. The 18-hour Brefeldin A resulted in increased detection of frequency of CD4+ T cells producing IFN-γ, IL-2, and TNF and combined cytokine producers. Five-day antigen stimulation improved the detection of IL-2, but not IFN-gamma or TNF as compared to the one day. In conclusion, detection of Mtb-specific T cell response using ICS of human PBMC is optimum using 1 day of antigen stimulation with additional Brefeldin A at the last 18 hours. The duration of antigen stimulation and protein transport inhibition affects the frequency and the functionality of cytokines analysed using flow cytometry. |
| Databáze: | OpenAIRE |
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