Nkp46 9e2

The following antibodies from BioLegend were used for flow cytometry: anti-human CD2 (TS1/8), CD3 (HIT3a), CD10 (HI10a), CD33 (WM53), CD34 (581), CD36 (5–271), CD38 (HB-7), CD45 (H130), CD45RA (HI100), CD56 (HCD56), CD94 (DX22), CD122 (TU27), CD132 (TUGh4), CD135 (BV10A4H2), NKG2D (1D11) and NKp46 (9E2). Anti-human NKG2A antibody was from R&D systems (Minneapolis, MN). Anti-mouse CD45 (30-F11) was acquired from BD Biosciences. For flow cytometry, samples were stained with antibodies in 50 μl flow buffer (0.2% bovine serum albumin [BSA, sigma], 0.05% sodium azide [sigma] in PBS) on ice for 30 mins. Stained samples were then analyzed on a BD LSR II flow cytometer, and data analyzed with FACS Diva (BD Biosciences) and FlowJo (TreeStar version e.g. 7.6.5). Isotype-matched control antibodies were used for all fluorochrome-isotype combinations. For fluorescent-activated cell sorting (FACS), cells were stained with appropriate antibodies in RoboSep buffer (Stemcell Technologies), and sorted on a BD FACSAria II (BD Bioscience).

For surface marker staining, NK cells were washed with PBS and stained with FITC, PE- and PerCP-conjugated mAb for 20 min; the mAb anti-human CD56 (NCAM16.2), CD16 (B73.1), CD158a (HP-3E4), Nkp44 (p44-8), CD8 (SK1) (all from BD Biosciences, San Jose, CA, USA), CD159a (131,411 purchased from R&D Systems, Minneapolis, MN, USA), NKG2D (1D11) and NKp46 (9E2) (all purchased from Biolegend, San Diego, CA, USA) and TRPA1 (Alomone, Israel) were used followed by anti-rabbit PE (R&D) as a secondary antibody. Mouse IgG isotypes were used as controls (BD Biosciences). For acquisition and analysis, the first was performed using an Attune Nxt (Life Technologies, Carlsbad, CA, USA) cytofluorimeter whereas the second was performed using Flow logic software 7.1 (Miltenyi), according to guidelines for the use of flow cytometry and cell sorting in immunological studies [41 (link)].