Preparation of Eight-Chambered Glass Coverslips for NK Cell Binding Studies

Preparation of eight-chambered glass coverslips was adapted from published protocols (Culley et al., 2009 (link)). Briefly, slides (1.5 Lab-Tek II; Nunc) were coated with 0.01% poly-L-lysine, dried, and coated with ligands for NK cells in PBS overnight at 4°C. 2.5 µg/ml MICA (Fc tagged; R&D Systems), 2.5 µg/ml MICA (His-tagged; Acro Biosystems), 10 µg/ml rituximab (GlaxoSmithKline), 2.5 µg/ml ULBP2 (Fc tagged; Abcam), 10 µg/ml anti-CD16 (clone 3G8; BioLegend), or 10 µg/ml anti-NKp30 mAb (clone P30-15; BioLegend), all with 2.5 µg/ml ICAM-1 (not tagged; R&D Systems) or 2.5 µg/ml Noggin (Fc tagged; R&D Systems), were used unless indicated otherwise. Where noted, 2.5 µg/ml ICAM-1 alone or 2.5 µg/ml of Noggin alone were also used as controls. In all experiments, recombinant human proteins MICA, ULBP2, and Noggin were used as Fc constructs and are referred throughout as MICA, ULBP2, and Noggin unless indicated otherwise. Slides were then washed with PBS and used for sample preparation.