Proliferation assay

PBMCs or enriched NK cells (see Isolating NK cells from human blood products ) are labeled with a permanent amine-reactive dye (CellTrace Violet Proliferation Kit, Cat. No:C34557, Thermo Fisher) according to the manufacturer's instructions.

Cells are exposed to various experimental conditions, then harvested after 7 days of culture.

Optional: If required, cells can be stained for apoptotic markers (FITC Annexin V, Cat. No: 556419, BD Biosciences) according to the manufacturer's instructions prior to antibody staining.

Cells are stained with a dead cell marker (LIVE/DEAD NEAR-IR, Cat. No: L34976, Thermo Fisher), PE-CY7 conjugated anti-CD56 (HCD56, BioLegend), and PE-CF594 conjugated anti-CD3 (UCHT1, BD BioSciences).

The samples are run on a flow cytometer (LSRII, BD Biosciences) and analyzed using FlowJo software (Tree Star Inc., RRID:SCR_008520).

The proliferation of live (dead cell marker- and annexin V-) NK cells (CD56+/CD3-) and T cells (CD56-/CD3+) is assessed by dilution of the dye.

Relative numbers of live NK cells and T cells for each treatment condition are obtained by running the flow cytometer at a constant flow rate and analyzing the number of each cell type obtained within a specified time limit e.g. 60s.