Goat anti mouse igg alexfluor 647

FACS binding experiments were performed with CHP-134 wild-type and CRISPR knockout clone cells as described previously [22 (link)]. Cells were suspended in fresh RPMI 10% FBS medium. The cell count was adjusted to 10⁶ live cells/mL. Test Abs and controls were added (10 μg/mL) to cells in tubes and incubated at ambient temperature for 45 min while mixing by mechanically rotating the tubes. The cells were washed with RPMI 10% FBS, then resuspended in the same medium containing secondary goat anti-mouse IgG AlexFluor 647 (Thermo Fisher Scientific). After 30 min incubation with mixing, the cells were washed once with Dulbecco’s phosphate buffered saline (DPBS) without Mg²⁺ or Ca²⁺ salts and suspended in DPBS containing 0.5% (volume/volume) formaldehyde for 10 min at room temperature. Binding was analyzed using a LSR Fortessa Flow Cytometer (BD Biosciences, San Jose, CA). FlowJo (TreeStar, Woodburn, OR) was used for data analysis.

Glycan array analysis was performed using an NHS ester-coated glass microarray slide containing six replicates of 130 diverse sialic acid-containing glycans, including terminal sequences and intact N-linked and O-linked glycans found on mammalian and avian glycoproteins and glycolipids^{65 (link)}. Recombinant HAs (50 μg mL⁻¹ final) were expressed and purified as described previously^{65 (link)}, before precomplexing with monoclonal mouse anti-His-Tag IgG (MA1-21315; Thermo Fisher Scientific) and goat anti-mouse IgG-Alex Fluor 647 (A-21235; Thermo Fisher Scientific) antibodies for 30 min at RT in 1× PBS-T. HA-antibody complexes were incubated on the array surface for 1 h at room temperature. Slide scanning to detect bound HA was conducted using an Innoscan1100AL (Innopsys) fluorescent microarray scanner. Fluorescent signal intensity was measured using Mapix (Innopsys) and mean intensity minus mean background of 4 replicate spots was calculated. A complete list of the glycans on the array is presented in Supplementary Fig. 12.