A flow-cytometry-based assay to assess granule exocytosis and GZB delivery by human CD8 T cells and NK cells

• Published in: STAR protocols 2023-03, Vol.4 (1), p.101939-101939, Article 101939
• Main Authors: Li, Tong, Smith, Mindy, Abdussamad, Maryam, Katz, Grace, Catalfamo, Marta
• Format: Article
• Language: English
• Subjects: Cell Biology; Cell-based Assays; Flow Cytometry/Mass Cytometry; Immunology; Protocol
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101939

CD8 T and NK cells mediate killing by delivery of perforin and granzyme B (GZB) stored in lysosome-like granules. We present a flow-cytometry-based protocol combined with a redirected killing assay to evaluate granule exocytosis and the cytotoxic potential of human CD8 T cells and NK cells. We describe the assessment of the delivered GZB inside the target cells. We then detail the detection of lysosome membrane protein CD107a exposed on the cell surface of the effector cells upon degranulation. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).1 [Display omitted] •Flow cytometry protocol to evaluate GZB delivery and activity inside the target cells•Redirected killing assay to assess the full cytotoxic potential of CD8 T cells•Protocol for the evaluation of the ex vivo cytotoxicity of human NK cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. CD8 T and NK cells mediate killing by delivery of perforin and granzyme B (GZB) stored in lysosome-like granules. We present a flow-cytometry-based protocol combined with a redirected killing assay to evaluate granule exocytosis and the cytotoxic potential of human CD8 T cells and NK cells. We describe the assessment of the delivered GZB inside the target cells. We then detail the detection of lysosome membrane protein CD107a exposed on the cell surface of the effector cells upon degranulation.