T cells and NK cells deploy potent immune defenses through the release of lysosomal granule contents such as granzymes and perforin. Lysosomes containing cytotoxic granules move to the cell surface and fuse with the plasma membrane to release their contents upon specific receptor engagement between a target cell and cytotoxic cell. This lysosomal fusion results in lysosomal-associated membrane glycoproteins (LAMPs), including CD107a (LAMP-1) and CD107b (LAMP-2), to be presented on the cell surface.
Methods for measuring cytotoxic cell degranulation were first developed in the 1960’s and include the chromium release assay, which is still widely used and is relatively easy to carry out, but has fallen out of favor because radioactive chromium is used.[1] Non-radioactive methods have also been developed, including ELISpot assays that measure specific molecules like IFN-gamma.
Staining for cell surface expression of CD107a and b has emerged as a leading method for measuring degranulation by flow cytometry[2]. Anti-CD107a and b staining can be added to an existing staining panel, although CD107 expression is notoriously transient, so refer to specific CD107 protocols if you plan to amend your existing staining protocol. Intracellular staining protocols can also be used to detect degranulation effector molecules such as perforin, granzymes, and granulysin by using methods that retain the contents of these lysosomes within the cell.
A flow cytometry-based degranulation assay can be done alongside a chromium release or ELISpot assay in order to gain insight into degranulation and target cell killing levels. Consider measuring degranulation as you plan to characterize effector cell functions in your next experiment.
[1] Zaritskaya L, Shurin MR, Sayers TJ, Malyguine AM. New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert Review of Vaccines. 2010. 9(6):601-616.
[2] Betts MR1, Brenchley JM, Price DA, De Rosa SC, Douek DC, Roederer M, Koup RA. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. 2003. J Immunol Methods. Oct 1; 281(1-2):65-78.
