Anti nkp30 p30 15

Cells were analyzed using a BD LSR Fortessa™ flow cytometer following staining with fluorochrome conjugated antibodies. Prior to staining, cells were fixed using the fix buffer set (Biolegend), according to the manufacturer’s instructions. Antibodies used within this study were conjugated anti-CD3 (UCHT1, Biolegend), anti-CD56 (N901, Beckman Coulter), anti-NKp30 (P30-15, Biolegend), anti-NKp44 (P44-8.1, BD), anti-NKp40 (29A1.4, Biolegend), anti-TNF-α (Mab11, eBioscience), anti-IFN-γ (485.B3, Biolegend). Annexin V/PI staining was performed according to the manufacturer’s protocol (Biolegend). The data was analyzed using FlowJo 10.7.1 software.

NK cells killing experiments were performed as previously described (20 (link)). Briefly, 1 × 106 NK cells were stimulated with IL-15 in the presence of DMSO, JQ1(+) or AZD5153 for 24 h. The cells were washed twice in fresh medium before being added to the NK cell killing assay. In a 96-well round bottom plate 1 × 105 NK cells at an effector-target ratio of 5:1 for 4 h. Target cells were JURKAT (clone E601, ATCC) and K562 (clone CCL-234, ATCC). Lactate dehydrogenase release was measured in 50 µl of supernatant using the Cytotox 96 assay kit (Promega), according to manufacturer’s instructions.
In parallel, NK cell degranulation assay was performed as previously described (21 (link)). Briefly, pre-treated NK cells were cultured with target cells at a 1:5 ratio in the presence of anti-CD107a-Fitc antibody (H4A3, Biolegend) and incubated for 1 h at 37°C. Protein transport inhibitor (eBiosciences) was added for the final 3 h of culture. After staining with anti-CD56, Anexin V and PI, the sample was assessed by flow cytometry on a BD LSR Fortessa™ instrument. For blocking antibody studies anti-IgG1 (12G8G11, BioLegend), anti-NKp30 (P30-15, BioLegend), NKp-44 (P44-8.1, BD), and NKp-46 (29A1.4, BioLegend) were added at the beginning of the assay. Data analysis was performed using the FlowJo program.