Human igg1 isotype control

1 × 10⁵ tumor cells per well were cultured in 96-well plates (flat bottom plate for adherent cells, round bottom plate for suspension cells) at 37 °C 5% CO₂. 7C6 (a human IgG1 mAb kindly provided by Dr Kai W. Wucherpfennig) or human IgG1 isotype control (Biolegend, Koblenz, Germany) antibody were added at 10 µg/mL. After 24 h of culture, MICA and MICB on cell surface were detected following staining with APC conjugated anti-MICA/B antibody or IgG2a isotype control. For detaching adherent cells without disturbing the integrity of surface molecule, Accutase (Biolegend, Koblenz, Germany) was used. Prior to the staining process, Fc receptors were blocked with Human TrueStain FcX™ (Biolegend, Koblenz, Germany) at a final dilution of 1:100. Hoechst 33258 (Cayman Chemical, Hamburg, Germany) was added before flow cytometry analysis for viable cells gating. Samples were acquired using FACS Canto II (BD).

Sub-confluent Vero cells in 24-well plates were infected in duplicate with ten-fold serially diluted viruses. After 2 h incubation at 37 °C, cells were washed with Opti-MEM (Thermo Fisher Scientific) and overlaid with 1 ml per well of 0.8% methylcellulose dissolved in Opti-MEM containing 1% L-glutamine, 2.5% penicillin-streptomycin, 0.1% gentamicin, 0.5% amphotericin B and 2% TrypLE Select (Thermo Fisher Scientific). On day 4 post-infection, cells were fixed using ice-cold 80% methanol. For dual-staining assays to detect expression of APMV3 and SARS-CoV-2 antigens, wells were incubated for 2 h at room temperature (RT) with a primary chicken anti-APMV3 serum (1:2,000) and a human anti-SARS-CoV-2 S RBD antibody (CR3022, 1:2,000) (or, as a negative control, was replaced with a combination of normal chicken IgY control (R&D Systems, cat# AB-101-C, 1:1,000) and human IgG1 isotype control (BioLegend, cat# 403501, 1:1,000)). After extensive washing with PBS, wells were incubated for 1 h at RT with 1:2,000 diluted infrared dye (IRDye)-conjugated 680LT donkey anti-chicken IgY (LI-COR, cat# 926-68028) and IRDye 800CW goat anti-human IgG secondary antibodies (LI-COR, cat# 926-32232). After washing with PBS, plates were scanned with an Odyssey CLx imager (LI-COR).