Anti cd69 bv650

Cells were stained for surface markers for 20 min at room temperature in PBS (Sigma). Extracellular antibodies used were as follows: LIVE/DEAD Fixable Near-IR (Thermo Fischer Scientific; L10119), Anti-CD3-BUV737 (BD Biosciences; 612750), Anti-CD14-BUV737 (BD Biosciences; 612763); Anti-CD14-BUV395 (BD Biosciences; 563561); Anti-CD19-BUV737 (BD Biosciences; 564303), Anti-CD19-BUV395 (BD Biosciences; 563549); Anti-CD56-PerCP-Cy5.5 (Biolegend; 304626), Anti-CD16-Pe-Cy7 (Biolegend; 302016), Anti-CD107a-BV510 (Biolegend; 328632), Anti-CD69-BV650 (Biolegend; 310934). Samples were then washed and fixed using 4% formaldehyde or BD Cytofix/Cytoperm Kit (BD biosciences) according to manufacturer’s directions. Flow cytometry analysis was performed on a LSRII instrument (BD Biosciences). A total of 50,000 to 250,000 events were acquired and analyzed using FlowJo software. The analysis was performed on gated cells that fell within the lymphocyte population, stained as live cells using Live/Dead stain. Cells were then gated for negative CD3/CD14/CD19 expression to exclude other cell populations, and NK cells were identified using CD56/CD16 gating strategy. Gating strategies are shown in Supplementary Figure S1. Within the NK cell population, we analyzed expression of CD107a and CD69 for each sample compared to their corresponding unstimulated control.

Samples were incubated with antibodies diluted in PBS containing 0.2% FCS (FACS buffer) for 30 min at 4°C before samples were analyzed using a Fortessa X20 (BD Biosciences) and analysis was performed using FlowJo software (Becton Dickinson). Gating strategy is shown in Supplementary Figure 1. Antibodies used include anti-MYCTag-AF488 (Clone 9B11, Cell Signaling Technology), anti-CD25-APC (Clone PC61, BioLegend), anti-CD69-BV650 (Clone H1.2F3, BioLegend), anti-CD4-FITC (Clone GK1.5, produced at WEHI Bundoora), and anti-CD8-PE antibodies (Clone 53-6.7, BD Pharmingen).