Anti cd107a pe cy5 clone h4a3

PBMCs or purified NK cells were stimulated with P815 mouse mastocytoma cells plus P815-specific Abs (1:100 dilution of polyclonal rabbit anti-mouse lymphocyte serum, Accurate Chemical & Scientific Corp., Westbury, NY) and ADCC responses were indicated through CD107a production and IFN-γ secretion from activated NK cells. P815 cells were used as target cells (18 (link)). PBMCs or purified NK cells were stimulated with P815 cells alone or Ab-opsonized P815 cells at an E: T ratio of 10:1. Then 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 6 h in a humidified CO₂ incubator at 37°C. Cells were stained with anti-CD3 eFluor 450 (clone UCHT1) and anti-CD56 PE-Cy7 (clone B159) and fixed by 2% 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).