Cd16 percp cy5

This study utilized frozen PBMC that were previously collected using the Ficoll-Paque method and stored in liquid nitrogen
[24 (link)]. PBMC were thawed in a water-bath at 37°C for one minute and thereafter, washed and re-suspended in RPMI media containing 10% v/v fetal calf serum before surface staining. PBMC viability was evaluated using trypan blue dye and viable cells were counted under microscopy. Minimum cell viability was 80%. Cells were rested for four hours, and subsequently stained with the following anti-human monoclonal antibodies; CD14/19 FITC CD3 Amcyan, CD8 APC-Cy7, CD16 PerCP Cy5.5, CD56 PE Cy7, NKG2D PE and NKp46 APC (BD Biosciences, San Jose CA). At least 50,000 events in the CD3-negative gate were collected. Gating was standardized and set using fluorescence minus one control (FMOs) for CD14/19, CD16, CD56, NKG2D and NKp46. NK cells were identified as CD3^-negative, CD14^-/CD19^- and NK cell subsets were identified by co-expression of CD56 and CD16 on NK cells; CD56^bri (CD56⁺⁺CD16^-), CD56^INTERMEDIATE (CD56⁺CD16^-), CD56^dim (CD56⁺CD16⁺) and CD56^neg (CD56^-CD16⁺); see Figure 
2. Expression of NK receptors NKG2D and NKp46 was defined by the percentage of NKG2D + and NKp46+ NK cell subsets (Figure 
3).

We used the following antibodies in this study: NKG2C-APC, NKG2C-PerCp, NKG2A-PE (R&D); CD3-FITC, CD4-APC-Cy7, CD16-PerCP-Cy5.5, CD56-PE-Cy7, CD107a-APC-H7 (BD Biosciences, San Jose, CA, USA); IFN-γ-BV421 (Biolegend, San Diego, CA, USA); CD4-FITC/CD8-PE/CD3-PerCP (BD Biosciences, San Jose, CA, USA).

Antibodies against Citrullinated Histone H3 (ab5103), PAD2 (ab16478), PAD4 (ab128086), PAD3 (ab183209), Tubulin and β-actin (ACTN05) were purchased from Abcam (Cambridge, UK). The anti-peptidyl-citrulline antibody, clone F95 (MABN328) was from EMD Millipore (Hertfordshire, UK). Antibodies against total histone H3 (1B1B2) and (96C10) were from Cell Signaling Technologies (Hertfordshire, UK). PAD1 (HPA062294) was from Sigma Aldrich (Dorset, UK), and PAD2 (12110-1-AP) was from Proteintech (Manchester, UK). All secondary antibodies used were raised in goat. F(ab')2 anti-Rabbit IgG A647, F(ab')2 anti-Mouse IgG PE (A10543), anti-Mouse IgG A594 (A11032) and anti-Mouse IgG A488 (A11029) were from Thermo Fisher Scientific (UK). Anti-Mouse IgM Texas Red was from VectorLabs (Peterborough, UK) and Anti-Rabbit IgG FITC was from Sigma Aldrich (Dorset, UK).
For flow cytometry, the following mouse anti human antibodies CD3 PEcy7, CD16 PerCP-Cy 5.5, CD19 BV786, CD56 BV650, and CD14 PE were obtained from BD Biosciences (Oxford, UK).

PBMCs from healthy human donors were added to 24-well plates, either alone or into wells that contained SN12C cells that had been plated 24 hrs previously. The cultures were incubated with enavatuzumab or a control antibody (10 μg/mL) for 24 hrs, after which the immune cells were removed to measure activation markers by flow cytometry. Immune cells were stained for monocytes and nature killer (NK) cells with fluorochrome-conjugated antibodies purchased from BD Biosciences: CD3-FITC, CD54-PE, CD16-PerCP Cy5.5, CD14-PE Cy7, CD56-APC, and CD69-APC Cy7. In some experiments, tumor cell cytotoxicity was assessed after 24 hr culture by measuring the level of cytokeratin18 in the supernatant by M65® ELISA (Peviva, Bromma, Sweden), according to the manufacturer's instructions.