Control igg

Isolation of human neutrophils from peripheral blood was carried out by using Ficoll-Paque PLUS density gradient (GE Healthcare) according to the procedures described previously [51 (link)]. ADCP by THP1 cells (from ATCC) and neutrophils was performed essentially as a previous report [52 (link)]. Control IgG was purchased from GeneTex (GTX16193). Briefly, Cal/09 virus was incubated with indicated antibodies at 37°C for 1 h and added to the sialidase (0.5 unit/mL, Sigma) pre-treated THP-1 cells or neutrophils. After 1 h incubation, THP-1 cells or neutrophils were washed three times with RPMI and incubated at 37°C. After 6 h incubation, THP-1 cells or neutrophils were fixed in 4% paraformaldehyde, permeabilized and stained with anti-influenza NP antibody (Abcam, Ab20921, 1:100 dilution). The levels of phagocytosis were monitored by flow cytometry.

ADCC was performed as previously reported [44 (link)]. HEK293T cells transfected with HA-expression vectors (Bris/07, Cal/09, H3, and H7) for 48 h and human peripheral blood mononuclear cells (PBMCs) were used as target cells and effector cells, respectively. Human PBMCs from healthy donors were obtained from Taipei Blood Center with the consent procedures approved by the Academia Sinica Research Ethics Committee. PBMCs were isolated by density gradient centrifugation with Ficoll-Paque at 400×g for 30 min without brake at 22°C. Control IgG was purchased from GeneTex (GTX16193). mAbs at 2-fold serial dilutions were added to the co-culture composed of 5×10³ 293T cells transiently expressing HA proteins from indicated strains of influenza viruses and 2.5×10⁵ effector PBMCs (E:T ratio = 50), and incubated for 5 h at 37°C. The supernatant of the co-culture was collected and analyzed by CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega).