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. 2023 Dec 15;4(4):102584.
doi: 10.1016/j.xpro.2023.102584. Epub 2023 Sep 21.

FluoroSpot assay to analyze SARS-CoV-2-specific T cell responses

Camilla Tiezzi  1 , Marzia Rossi  1 , Andrea Vecchi  2 , Sara Doselli  2 , Amalia Penna  2 , Paola Fisicaro  2 , Ilaria Montali  1 , Camilla Ceccatelli Berti  1 , Valentina Reverberi  2 , Anna Montali  1 , Gabriele Missale  3 , Carlo Ferrari  3 , Carolina Boni  4
Affiliations

FluoroSpot assay to analyze SARS-CoV-2-specific T cell responses

Camilla Tiezzi et al. STAR Protoc. .

Abstract

Monitoring antigen-specific T cell frequency and function is essential to assess the host immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we present a FluoroSpot assay for concurrently detecting ex vivo antiviral cytokine production by SARS-CoV-2-specific T cells following peptide stimulation. We then detail intracellular cytokine staining by flow cytometry to further validate the FluoroSpot assay results and define the specific T cell subpopulations. For complete details on the use and execution of this protocol, please refer to Tiezzi et al. (2023).1.

Keywords: Cell Biology; Flow Cytometry/Mass Cytometry; Immunology.

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Conflict of interest statement

Declaration of interests C.F. has received grants from Gilead and AbbVie and is a consultant at Gilead, AbbVie, Vir Biotechnology Inc, Arrowhead, Transgene, and BMS.

Figures

None
Graphical abstract
Figure 1
Figure 1
Results of FluoroSpot assay from a representative vaccinated subject (A) Example of raw data derived from the plate reader. Red numbers indicate positive spots. (B) Image of IFN-γ, TNF-α and IL-2 production exported from the ImmunoSpot 7 Software.
Figure 2
Figure 2
Gating strategy for the intracellular cytokine staining performed with PBMC stimulated by peptide pools
See this image and copyright information in PMC

References

    1. Tiezzi C., Vecchi A., Rossi M., Cavazzini D., Bolchi A., Laccabue D., Doselli S., Penna A., Sacchelli L., Brillo F., et al. Natural heteroclitic-like peptides are generated by SARS-CoV-2 mutations. iScience. 2023;26 doi: 10.1016/j.isci.2023.106940. - DOI - PMC - PubMed

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