Fitc conjugated cd107a

TLR activation of NK cells was analysed concerning induction IFN-gamma and TNF-alpha as well as CD107a degranulation in response to K562 target cells. Purified NK cells were pre-activated overnight with IL-2 and then cultured with and without Pam3CSK4, LPS-B5 and CpG-ODN-2216, respectively. After 16 h, NK cells were divided into either co-culture with K562 effector cells (effector to target ratio of 1:2) or kept in culture with medium alone. Next, FITC-conjugated CD107a (BD Biosciences, Heidelberg, Germany) was added. After 1 h, Golgi Stop (BD Biosciences) and BFA (Enzo Life Sciences GmbH) were added for additional 3 h. Then, cells were stained with Zombie AquaTM (BioLegend, London, UK) followed by staining with anti-CD3 (APC-Cy7-labelled), anti-CD56 (Brilliant Violet 421-labelled) and anti-CD16 (PerCP-labelled). After fixation and permeabilization, cells were stained intracellularly with anti-IFN-gamma (PE-labelled) and anti-TNF-alpha (PE-Cy7-labelled) (all BioLegend). Percent IFN-gamma-, TNF-alpha- and CD107a-positive CD56^dimCD16^pos, CD56^dimCD16^neg and CD56^highCD16^neg NK cells were measured before and after TLR stimulation as well as after co-culture of NK cells with K562 target cells in the presence and absence of TLR stimulation (Supplementary Figure 2).

The cells were surface-stained with fluorochrome-conjugated Abs for 20 min on ice using the following Abs: anti-CD3, anti-CD4, anti-CD8, anti-NK1.1, anti-CD69 (eBioscience), and anti-Foxp3 (BioLegend, San Diego, CA, USA). To perform intracellular cytokine staining, IHLs were co-cultured with SL4 cells (1 × 10⁵ cells/well) for 4 h at 37°C in 96-well round-bottom plates containing 200 μL/well RPMI-1640 medium. Mouse recombinant IL-2 (50 units) and 0.2 μL of BD GolgiPlug protein transport inhibitor (BD Biosciences) were added to each well. After incubation, the cells were harvested, washed in PBS containing 1% FBS, and incubated for 10 min on ice with unlabeled anti-mouse CD16/32 Ab (BD Biosciences) to block FcγRII/III binding. The cells were then surface-stained for 20 min on ice with the indicated Abs. Following staining, the cells were washed to remove unbound Ab and fixed using a Cytofix/Cytoperm Kit (BD Biosciences). Cells were then subjected to secondary staining with reagents obtained from BioLegend except as noted, including the following: FITC-conjugated CD107a, PE-conjugated anti-interferon gamma, and anti-IL-10 (BD Biosciences). Data were acquired using a FACSCalibur flow cytometer and analyzed using CellQuest (BD Immunocytometry Systems, San Jose, CA, USA) and FlowJo software (Tree Star, San Carlos, CA, USA).