Attune focusing cytometer

Natural killer cytotoxicity and IFN-γ production were analyzed in co-cultures of primary NK cells and K562 cells (an NK-sensitive target cell line that lacks MHC-I molecules) with and without previous exposure of the NK cells to individual LRAs from our panel. Cytotoxicity was assessed by analyzing the expression of the degranulation marker CD107a, a reliable marker of NK cell cytotoxic activity (28 (link)). A total of 100,000 NK cells were co-cultured with the same number of K562 target cells in 96-well plates for 4–6 h in the presence of PE/Cy7-CD107a antibody, clone H4A3 (BD), adding 1 μL of GolgiStop (BD) after the first hour of culture. Cells were then harvested, washed, and surface-stained with CD56-FITC, clone NCAM 16 (BD) in staining buffer for 20 min on ice in the dark. Cells were then fixed with Fixation buffer (Biolegend, San Diego, CA, USA) during 20 min at room temperature in the dark, washed with Perm/Wash buffer twice and intracellularly stained with IFNγ-PE (Biolegend, San Diego, CA, USA) for 20 min. After washing, cells were re-suspended in staining buffer and analyzed in the Attune Focusing Cytometer (Applied Biosystems). To analyze whether NK cells were non-specifically activated by LRA, NK cells were also incubated in the absence of target cells, and CD69 (CD69-PE, clone FN50, from BD) and CD107a expression was analyzed as described.

Natural killer cells were cultured in cIMDM in the presence or absence of the individual LRAs from our panel for 24 h. After washing, cells were re-suspended in Annexin binding buffer and stained with Annexin V-FITC and 7-AAD (Biolegend, San Diego, CA, USA) following manufacturers’ protocol. Samples were analyzed on the Attune Focusing Cytometer (Applied Biosystems), and the percentage of double-positive cells for both Annexin V and 7-AAD was considered as the non-viable population.

Harvested cells were stained with zombie yellow (for dead cells) then fixed with 4% paraformaldehyde. For SSEA1 detection, cells were incubated with primary antibody in 2% BSA–PBS. For other antibodies, cells were permeabilised and blocked for 15 min in FACS buffer (5% horse serum and 0.5% tween 20 in PBS) before being incubated with primary antibody in FACS buffer. Primary and secondary antibodies were the same as for immunofluorescence as described above. NSCs derived from E14 ES cells were utilised as a negative control for the detection of pluripotency and endodermal markers. FACS analysis was carried using the Attune Focusing Cytometer (Applied Biosystems, Thermo Fisher Scientific). The yellow-stained cells were excluded from the data.