Human IgG1 Fc (residues 216–447) was expressed using the HEK293F cells grown in Freestyle293 medium (Life Technologies) as previously described 21 (link). Afucosylated IgG1 Fc was expressed using culture medium supplemented with 250 μM 2-deoxy-2-fluoro-l-fucose (Santa Cruz Biotech)45 (link). Plasmids encoding the CD16a variants Asn45Q, Asn162Q, Asn45Q/Asn162Q, N38Q/N74Q/N169Q were prepared according to the QuickChange protocol (Agilent Technologies) and confirmed by DNA sequencing (ISU DNA facility). CD16a (residues 19–193, V158 allotype) was expressed using the HEK293F or HEK293S(lec1−/−) cell lines with Freestyle293 medium (Life Technologies) as previously described 21 (link), 39 . CD16a with Man9 oligomannose N-glycans was expressed by supplementing the expression medium with 5 μM kifunensine (Cayman Chemical). Following purification, proteins were exchanged into 20 mM 3-morpholinopropane-1-sulfonic acid (MOPS), 100 mM sodium chloride, pH 7.2. CD16a was stored at −80 °C in 25% glycerol (v/v). 13C labeled protein was prepared by supplementing the expression medium with [13C6]-d-glucose as previously described 39 . CD16a N-glycans were analyzed following derivatization with procainamide and HILIC-ESI-MS as previously described 24 (link).