Ghost violet 540 cell viability dye

Sorting was performed on a FACS Aria II cell sorter (BD). Cells of interest were harvested from differentiation cultures, and resuspended in MACS buffer (5% FBS, Gibco; 0.5 mM EDTA in PBS). The following antibodies were used: CD41a-APC, CD235a-PE, CD43-PerCP, CD45-PerCP, CD34-FITC (BD Pharmingen), and CD56-APC (BioLegend). Dead cells were counterstained with Ghost Violet 540 cell viability dye (TONBO Biosciences). Sorting gates were set with appropriate Fluorescent Minus One (FMO) controls, and only live cells were collected.

For assessment of intracellular perforin production, NK cells were incubated with Ghost violet 540 cell viability dye (TONBO Biosciences) and CD56-PerCP (BioLegend). After washing with MACS buffer, cells were then fixed and permeabilized, following instructions in the Intracellular Fixation and Permeabilization Buffer Set (Thermo Fisher). Cells were then incubated overnight with Perforin-FITC (BD Pharmingen), and analyzed by flow cytometry.

To assess interferon gamma production, FAC-sorted NK cells were stimulated for 5 hours with PMA and ionomyocin (1:500) Cell Activation Cocktail (BioLegend), or alternately K562 tumor target cells at a 2:1 ratio. Breferidin A (1:1000; Thermo Fisher) was added at the beginning of the stimulation. NK cells that were not stimulated served as experimental controls. At the completion of incubation, all cells were washed with MACS buffer (5% FBS, Gibco; 0.5 mM EDTA in PBS), and incubated with Ghost violet 540 cell viability dye (TONBO Biosciences) and CD56-PerCP (BioLegend), for 30 minutes. Subsequently, cells were permeabilized and stained with IFNγ antibody.