Mouse igg1 κ clone x40

1 × 10⁵ purified CD56+ T cells from healthy controls were stimulated with 10 μg/ml of purified anti-CD16 antibody (clone 3G8, Santz Cruz biotechnology, Santa Cruz, CA, USA) or 10 μg/ml purified anti-CD32 antibody (clone 3D3, Santz Cruz biotechnology) or mouse IgG1(κ) (clone X40, BD Biosciences) isotype control for 30 min on ice. Cells were washed to remove unbound antibody and incubated with 10 μg/ml of goat anti-mouse IgG1 F(ab′)₂ for 5 h (Santz Cruz biotechnology, Santa Cruz, USA) at 37 °C. Cells were washed and stained with anti-CD3 Pacific Blue (clone UCHT1), anti-CD56 PE-Cy7 (clone B159) and anti-CD107a PE-Cy5 (clone H4A3) and fixed by 2% paraformaldehyde (PFA). All data were acquired on BD FACS Fortessa (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo software (Treestar, Ashland, OR, USA).

PBMCs or purified NK cells were stimulated with 10 μg/ml of anti-CD16 antibody (clone 3G8, Santz Cruz biotechnology, Santa Cruz, CA, USA) or mouse IgG1(κ) (clone X40, BD Biosciences) isotype control for 30 min on ice. Cells were washed to remove unbound antibody and incubated with 10 μg/ml of goat anti-mouse IgG1 F(ab′)₂ (Santz Cruz biotechnology, Santa Cruz, USA). The Brefeldin-A (10 μg/ml, Sigma, St. Louis, MO, USA), GolgiStop (5 μg/ml, BD Biosciences) and anti-CD107a PE-Cy5 (clone H4A3, BD Biosciences) were added to cell medium after 1 h incubation and continued to incubate for up to 5 h in a humidified CO₂ incubator at 37°C. Then, cells were washed and stained with anti-CD3 eFluor 450 (clone UCHT1) and anti-CD56 PE-Cy7(clone B159) and fixed by 2% paraformaldehyde (PFA). All data were acquired on BD FACS Fortessa (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo software (Treestar, Ashland, OR, USA).

The PBMC and spleen were thawed and resuscitated to assess the NK cell function by three stimulation regimens: (1) the NK cell stimulant phorbol myristate acetate (PMA) and ionomycin; (2) co-culture with NK-sensitive K562 cells or (3) CD16 Fc-receptor crosslinking. Specifically, in the PMA plus ionomycin stimuli group, PBMCs and single-cell suspensions were stimulated for 2 h in an R10 culture media containing PMA (100 ng/mL, Sigma-Aldrich) and ionomycin (1 µg/mL, Santa Cruz Biotechnology, Santa Cruz, CA, USA). For the K562 cells co-cultured group, PBMC (or single-cell suspension) and K562 cells were co-incubated at an E: T ratio of 10:1 in the R10 culture media. For CD16 cross-linking, 1 × 10⁶ PBMCs or single-cell suspensions from the spleen were stimulated with 10 μg/mL of the purified anti-CD16 antibody (clone 3G8, Santz Cruz Biotechnology) or mouse IgG1(κ) (clone X40, BD Biosciences) isotype control for 30 min on ice. The cells were washed to remove unbound antibodies and incubated with 10 μg/mL of goat anti-mouse IgG1F(ab’)2 for 5 h (Santa Cruz Biotechnology) at 37 °C. The unstimulated PBMCs (or single-cell suspension) group containing the medium alone was used as a control. All stimulated or unstimulated cells were washed and stained following standard methods, and the expression levels of CD107a and IFN-γ were measured by Flow Cytometry.