Apc conjugated anti human tnf α

Recombinant human IL-2 and IL-15 (PeproTech, Rocky Hill, NJ, USA) were used to expand NK cells from PBMCs. Allophycocyanin (APC)-Cy7-conjugated anti-human CD3, phycoerythrin (PE)-Cy7-conjugated anti-human CD56, PE-Cy5-conjugated CD56, and fluorescein isothiocyanate (FITC)-conjugated CD3 (eBioscience, San Diego, CA, USA) were used to measure NK cell purity (CD56+/CD3–). Pacific blue-conjugated anti-human CD16, FITC-conjugated anti-human CD57, APC-conjugated anti-human DNAM-1, peridinin chlorophyll protein complex (PerCP)-conjugated anti-human NKG2D, PE-conjugated anti-human NKG2C, APC-conjugated anti-human NKG2A, and PE-conjugated anti-human NKp30, PerCP-Cyanine5.5-conjugated anti-human NKp44, Pacific blue-conjugated anti-human NKp46 (eBioscience), PerCP-Cyanine5.5-conjugated anti-human KIR2DL1, FITC-conjugated anti-human KIR2DL2/3, and Pacific blue-conjugated anti-human KIR3DL1 (BD Biosciences, Franklin Lakes, NJ, USA) were used to measure expanded NK cell receptor levels. BV421-conjugated anti-human IFN-γ and APC-conjugated anti-human TNF-α (BD Biosciences) were used for intracellular staining, and PE-conjugated anti-human CD107a (BD Biosciences) was used as a surrogate marker for degranulation.

Recombinant human IL-2 and IL-15 (PeproTech, Rocky Hill, NJ, USA) were used to expand NK cells, and IL-21 (PeproTech) was used to stimulate NK cells at the beginning of the culture at various concentrations. Phycoerythrin (PE)-conjugated anti-human CD134 antibody (OX40 Ab, clone ACT35), and PE-conjugated anti-human CD252 antibody (OX40 Ligand Ab, clone ik-1) (BD Biosciences, San Jose, CA, USA) were used to measure the expression of OX40 receptor and its ligand on K562 and expanded NK cells during the culture. APC-Cy7-conjugated anti-human CD3, PE-Cy7-conjugated anti-human CD56, pacific blue-conjugated anti-human CD16, pacific blue-conjugated anti-human NKp46, PerCP-conjugated anti-human NKG2D, PerCP-conjugated anti-human CD8a, FITC-conjugated anti-human CD62L, FITC-conjugated anti-human CD57 (eBioscience, San Diego, CA, USA), FITC-conjugated anti-human CD3, APC-conjugated anti-human CD25, PE-conjugated anti-human CD69, APC-conjugated anti-human DNAM-1, APC-conjugated anti-human NKG2A, PE-conjugated anti-human NKG2C, PE-conjugated anti-human NKp30 (BD Biosciences) were used to evaluate the purity and surface expression of NK cell receptors. BV421-conjugated anti-human IFN-γ, APC-conjugated anti-human TNF-α (BD Biosciences) were used for intracellular staining, and PE-conjugated anti-human CD107a (BD Biosciences) was used as a surrogate marker of degranulation.