Protocol for flow cytometry immunophenotyping of human antigen-specific T cells by activation-induced marker and Th1 cytokine detection

Gianluca Rotta; Valentina Achille; Scott J Bornheimer; Melanie Duenas; Marco Fernandez; Arianna Gatti; Sergio Haro Giron; Irene Martinez Rio; Marta Massanella; Diego J Jiménez; Jorge Monserrat; Verena Pfeifer; Josefa Pichler; Barbara Prietl; Harald Sourij; Chiara R M Uras; Sara Vlah; Daniela Fenoglio

doi:10.1016/j.xpro.2024.103343

Protocol for flow cytometry immunophenotyping of human antigen-specific T cells by activation-induced marker and Th1 cytokine detection

STAR Protoc. 2024 Dec 18;6(1):103343. doi: 10.1016/j.xpro.2024.103343. Online ahead of print.

Authors

Gianluca Rotta¹, Valentina Achille², Scott J Bornheimer³, Melanie Duenas⁴, Marco Fernandez⁵, Arianna Gatti⁶, Sergio Haro Giron⁷, Irene Martinez Rio⁴, Marta Massanella⁸, Diego J Jiménez⁷, Jorge Monserrat⁷, Verena Pfeifer⁹, Josefa Pichler¹⁰, Barbara Prietl⁹, Harald Sourij¹¹, Chiara R M Uras¹², Sara Vlah¹³, Daniela Fenoglio¹⁴

Affiliations

¹ Becton, Dickinson & Company, 20161 Milan, Italy. Electronic address: gianluca.rotta@bd.com.
² Cytogenetics and Molecular Genetics Laboratory ASST Ovest Milanese - Legnano Hospital, 20025 Legnano, Italy.
³ Becton, Dickinson & Company, Milpitas, CA 95035, USA.
⁴ Becton, Dickinson & Company, 28522 Madrid, Spain.
⁵ Flow Cytometry Core Facility Germans Trias i Pujol Research Institute, 08916 Badalona, Spain.
⁶ Transfusion Center ASST Ovest Milanese - Legnano Hospital, 20025 Legnano, Italy.
⁷ Department of Medicine and Medical Specialties, University of Alcalá, 28801 Alcalá de Henares, Spain.
⁸ IrsiCaixa AIDS Research Institute, Germans Trias i Pujol Research Institute (IGTP), Can Ruti Campus, Badalona, Catalonia, Spain.
⁹ Center for Biomarker Research in Medicine GmbH, Graz 8010, Austria; Division of Endocrinology and Diabetology, Medical University of Graz, Graz 8036, Austria.
¹⁰ Becton, Dickinson & Company, Vienna 1200, Austria.
¹¹ Division of Endocrinology and Diabetology, Medical University of Graz, Graz 8036, Austria.
¹² Department of Experimental Medicine, Centre of Excellence for Biomedical Research, University of Genoa, 16132 Genoa, Italy.
¹³ Becton, Dickinson & Company, 20161 Milan, Italy.
¹⁴ Department of Internal Medicine, University of Genoa, 16132 Genoa, Italy; IRCCS Ospedale Policlinico San Martino, 16132 Genoa, Italy. Electronic address: daniela.fenoglio@unige.it.

PMID: 39705144
DOI: 10.1016/j.xpro.2024.103343

Abstract

Flow cytometry characterization of antigen-specific polyfunctional T cells is a valuable tool to study adaptive immunity. Here, we present a protocol for flow cytometry immunophenotyping of human antigen-specific T cells by activation-induced marker (AIM) and Th1 cytokine detection. We describe steps for preparing peripheral blood mononuclear cells (PBMCs) for stimulation followed by washing and staining PBMCs for flow cytometry. We then detail procedures for acquisition and analysis. This protocol has potential applications in the field of vaccine immunology and immuno-oncology. For complete details on the use and execution of this protocol, please refer to Altosole et al.¹.

Keywords: Cell Biology; Cell culture; Cell-based Assays; Flow Cytometry; Immunology.