Identification, sorting and profiling of functional killer cells via the capture of fluorescent target-cell lysate

Assays for assessing cell-mediated cytotoxicity are largely target-cell-centric and cannot identify and isolate subpopulations of cytotoxic effector cells. Here we describe an assay compatible with flow cytometry for the accurate identification and sorting of functional killer-cell subpopulations in co-cultures. The assay, which we named PAINTKiller (for 'proximity affinity intracellular transfer identification of killer cells'), relies on the detection of an intracellular fluorescent protein 'painted' by a lysed cell on the surface of the lysing cytotoxic cell (specifically, on cell lysis the intracellular fluorescein derivative carboxyfluorescein succinimidyl ester is captured on the surface of the natural killer cell by an antibody for anti-fluorescein isothiocyanate linked to an antibody for the pan-leucocyte surface receptor CD45). The assay can be integrated with single-cell RNA sequencing for the analysis of molecular pathways associated with cell cytotoxicity and may be used to uncover correlates of functional immune responses. Subpopulations of functional killer cells in co-culture can be identified and sorted by an assay that detects an intracellular fluorescent protein from lysed cells on the surface of the lysing killer cells.

Automated summary

This article describes a flow cytometry assay that accurately identifies and sorts functional killer-cell subpopulations in co-cultures. The assay uses the detection of an intracellular fluorescent protein from lysed cells on the surface of the lysing killer cells, and can be integrated with single-cell RNA sequencing to analyze molecular pathways associated with cell cytotoxicity. It may help uncover correlates of functional immune responses.