Facslyrics cytometer

Serum incubated NK-92 cells were labelled with Fix/Via Stain 700(BD 564,997), PE-Cy7 conjugated anti-CD56- (eBioscience 25–0567-42), Alexa flour 647 conjugated mouse anti-IDO1 (BD 566,648), FITC conjugated KIR2DL1 (BD 556,062), BV421 conjugated mouse anti-AhR (BD 565,791), BV510 conjugated NKG2D (BD 563,266). All antibodies were purchased from BD Biosciences Heidelberg, Germany. Cells were incubated with 5 µL of each antibody for 20 min and 30 min for extracellular and intracellular staining, respectively, in the dark at room temperature, then washed and resuspended in 200 μl PBS prior to analyse on flow cytometry. Labelled cells were directly analysed with BD FACSLyrics cytometer. Live and dead cells were identified by Fix/Via stain through forward and side scatter characteristics and gated electronically using FlowJo™ software. CD56 + lymphocytes were gated as NK cells. The surface markers were identified by gating for NKG2D + , KIR2DL1 + and intracellular markers by AhR + , IDO + . We used one intracellular (IDO) and one extracellular (NKG2D) unstained marker for fluorescence minus two (FM2) control to determine the negative population. Representative plots demonstrating gating strategy are given in Appendix Fig. 1 (Supplementary file 1). The methodology and parameters used for flow cytometry data acquisition are listed in Appendix 1 (Supplementary file 1).

The cytokines IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ were detected in supernatants from each stimulation assay condition with the Th1, Th2, and Th17 cytometric bead array (CBA) (BD Bioscience, Franklin Lakes, NJ, USA) in accordance with the manufacturer’s instructions. Data were acquired using the BD FACSLyrics cytometer (BD, San José, CA, USA) and BD FACSuite software Version 1.4. Then, the data were analyzed, employing FCAP Array software version 3.0 (BD San José, CA, USA).