Flow logic software 7

NK cells were incubated with K562 cells (ATTC) for 6 h at 37 °C. After 2 h incubation, brefeldin (1 μg/mL, BD Bioscience) was added for the last 4 h. Cells were collected and stained with surface antibodies for 20 min followed by Cytofix/Cytoperm (BD Biosciences) treatment and finally stained for 20 min with the indicated intracellular antibodies mAb in the presence of Perm/Wash solution (BD Biosciences). For acquisition, Attune Nxt (Life Technologies) cytofluorimetry was used whereas, for analysis, Flow logic software 7.1(Miltenyi) was used. CD107 (H4A3) and Granzyme (GB11) were purchased from BD Biosciences, Perforin (PRF1) was purchased from Biolegend, and mouse IgG isotype controls were purchased from BD Biosciences.

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)].